Patent Grant
9926610
Gold nanoparticle-based colorimetric and fluorimetric assays for detection of amplified and unamplified nucleic acids in clinical specimens from Acinetobacter, mycobacteria, staphylococcus, hepatitis B virus, human immunodeficiency virus, influenza virus, and West Nile Virus.
Gold nanoparticles or AuNPs exhibit a unique phenomenon known as Surface Plasmon Resonance, which is responsible for their intense red color in suspension. This color changes to blue upon aggregation of AuNPs. Several molecular assays are available for detection of microbial DNA, including DNA from bacteria like Acinetobacteria, staphylococci, and mycobacteria, and from viruses such as Hepatitis B Virus, Human Immunodeficiency Virus (HIV), Influenza A Virus, and West Nile Virus. Despite the high sensitivity and specificity of these methods, they are time-consuming, labor intensive, expensive, and require specialized equipment and thus are not suitable for use in many developing countries or for use in the field. Therefore, there is a great need to develop a low-tech assay for the direct detection of unamplified nucleic acids from microbial with acceptable sensitivity and specificity, short turn around time, and cost-effectiveness. Such an assay would have great utility in quickly and inexpensively characterizing microbes and controlling microbial pathogens in developing countries with limited resources and high infection rates, such as Egypt.
Nanoparticles have been recently proposed as promising tools to develop the next generation of diagnostic assays. Because of their unique properties and ability to interact with biomolecules on one-to-one basis, various nanoparticles show great promise to meet the rigorous demands of the clinical laboratory for sensitivity and cost-effectiveness, and can be used in the future in point-of-care diagnosis.
Gold nanoparticles (“AuNPs”) are spheres with a typical diameter of approximately 2-50 nm. They exhibit a unique phenomenon known as Surface Plasmon Resonance (SPR), which is responsible for their intense red color, and which changes to blue upon aggregation of AuNPs.
The addition of salt shields the surface charge on the AuNPs, which are typically negatively charged owing to adsorbed negatively charged citrate ions on their surfaces, leading to aggregation of AuNPs and a red-to-blue color shift. SPR is also responsible for the large absorption and scattering cross-sections of AuNPs which are 4-5 orders of magnitude larger than those of conventional dyes. These unique optical properties have allowed the use of AuNPs in simple and rapid colorimetric assays for clinical diagnosis offering higher sensitivity and specificity than current detection techniques. Suitable components and procedures for making gold nanoparticles are known in the art and are incorporated by reference to the articles cited herein.
Li et al. developed a colorimetric assay using unmodified citrate-coated AuNPs. This method is based on the property of single-stranded DNA (ssDNA) which adsorbs on citrate-coated AuNPs. This adsorption increases the negative charge on the AuNPs leading to increased repulsion between the particles, thus preventing aggregation. The adsorption of ssDNA on AuNPs occurs due to the fact that ssDNA can uncoil and expose its nitrogenous bases. The attractive electrostatic forces between the bases and the AuNPs allow adsorption of the ssDNA. On the other hand, double-stranded DNA (dsDNA) does not adsorb on AuNPs due to the repulsion between its negatively-charged phosphate backbone and the negatively-charged coating of citrate ions on the surfaces of the AuNPs. Therefore, when AuNPs are added to a saline solution containing the target DNA and its complementary unlabeled single-stranded polynucleotide, AuNPs aggregate (since the single-stranded polynucleotides are not free to stabilize the AuNPs) and the solution color changes to blue. However, in the absence of the target or the presence of a non-complementary target, the complementary single-stranded polynucleotides are free to stabilize the AuNPs thus preventing their aggregation and the solution color remains red. This method has been used to detect single nucleotide polymorphisms in PCR-amplified genomic DNA extracted from clinical samples. Moreover, based on the same principle, AuNPs are capable of quenching fluorescent dyes and this property has been used for detection of synthetic nucleic sequences with high sensitivity and selectivity. Shawky, et al., Clin. Biochem. 43:1163-1168 (2010) disclosed direct detection of unamplified hepatitis C virus RNA using unmodified gold nanoparticles. However, this method has not been proven or evaluated for microbes which contain more extensive and complex genomes or for clinical samples containing such microbes.
Bacteria
Mycobacteria
Mycobacterium tuberculosis
Tuberculosis (TB) is a contagious disease caused by the airborne pathogen Mycobacterium tuberculosis. The disease currently infects about one third of the world's population, with 8-9 million new infections annually; it claims a victim every 10 seconds. With an estimated death toll of 1.3 million victims in 2008 alone, TB claims more human lives every year than any other single pathogen. In Africa, HIV is the single most important factor contributing to the increase the incidence of TB since 1990 and TB became generally the leading cause of HIV-related deaths. The M. tuberculosis complex includes other TB-causing mycobacteria: M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti and M. pinnipedii. Mycobacterium bovis and Mycobacterium microti are also the causative agents of TB in animals but can be transmitted to humans, such as immunocompromised patients.
Mycobacterium tuberculosis is an acid fast beaded bacillus. It is non-motile non-spore forming rod which appears either straight or slightly curved under microscopic examination. Its genomic DNA has 70% GC content and its cell wall has high lipid content. It is able to survive in harsh environmental conditions and divides inside of host cells every 12-24 h. Once the TB bacilli are inhaled, they enter the alveoli to the alveolar space which is the first to have a role in eliminating the bacteria recruiting macrophages and pathogenesis in the lung starts. The first contact of the bacilli is with macrophage where they have the ability to reside in the macrophage bypassing phagocytosis and multiply but develop to dormant stage with low replication rate and metabolic activity.
The disease can develop to be extra-pulmonary, the infection proceed in other organs such as bones and brains. The bacilli cause high inflammatory host responses which may be fatal. The clinical manifestation starts with non specific symptoms including cough, fever, malaise, and weight loss. At late stages, bloody reproductive cough develop with pulmonary symptoms.
Nontuberculous Mycobacteria (NTM)
Nontuberculous mycobacteria (NTM) include the mycobacterial species other than those comprising the Mycobacterium tuberculosis complex. They are opportunistic environmental bacterial pathogens that commonly inhabit different water bodies and soil and have the ability to attach to surfaces and form biofilms. This accounts for their survival in areas with direct human contact such as water plumbing and pose a threat of nosocomial infection in case of adherence to areas such catheter surfaces. Nevertheless, there are no known instances of human-to-human transmission and environmental exposure is believed to be the path to infection. NTM are not obligate pathogens and their main threat is in the case of immunocompromised patients, e.g., in subjects having AIDS and cystic fibrosis, which accounts for their increased importance with the HIV pandemic. NTM members include slow-growing bacteria (colony appearance on solid media takes more than 7 days) such as M. ulcerans, M. kansasii, and M. scrofulaceum, and rapid-growing bacteria (colonies appear on solid media in ≤7 day) such as M. fortuitum, M. abscessus, and M. mucogenicum. Another notable NTM member is noncultivatable M. leprae, the causative agent of leprosy. NTM can cause various diseases including pulmonary infections with TB-like presentations e.g. M. avium and M. malmoense, skin and soft-tissue infections and post-operative wound infections, e.g. M. fortuitum and M. mucogenicum, in addition to gastrointestinal diseases, lymphadenitis, and disseminated disease in immunocompromised patients. Treatment of NTM is challenging as many members are resistant to most common anti-mycobacterial drugs and the treatment must be customized depending on the infecting species and the clinical manifestation. This makes their prompt diagnosis essential for effective patient management.
Acinetobacter
Acinetobacter is a genus of gram-negative bacteria associated with a variety of hospital-acquired infection. Their ability for long-term survival in hospital environment and extreme ability to develop resistance to all the known conventional antibiotics raised serious concerns throughout the past decades. Under microscopical examination, they are short, plump, gram negative rods that are difficult to destain and may therefore be misidentified as either gram negative or gram positive cocci.
Acinetobacters are strictly aerobic, non-fermenting and non-motile short gram-negative rods in the logarithmic phase becoming more coccoid at the stationary phase. Acinetobacters grow readily on common laboratory media and are usually isolated on blood agar.
Acinetobacter genus is classified into 33 species based on genetic relatedness. Species 2 (Acinetobacter baumannii) is the most important clinically, where about 90% of the clinical isolates are found to be infected with Acinetobacter baumannii.
A. baumannii is an opportunistic pathogen that infects immune-compromised patients. It is a causative agent of nosocomial pneumonia, bacteremia, meningitis, and urinary tract infection, resulting in attributable mortalities of up to 23% for hospitalized patients and 43% for patients under intensive care. More recently Acinetobacter has caused serious infections among American military personnel serving in Iraq and Afghanistan.
The reason why Acinetobacter infections pose such a serious problem is due to the bacteria's minimal nutritional requirements and their ability to survive under harsh conditions of pH & temperature. These properties facilitate Acinetobacter's ability to colonize inert surfaces such as hospital surfaces and tools for prolonged periods. Additionally, the bacterium has the ability to develop and express resistance to nearly all available antibiotics.
A. baumannii, along with Aspergillus spp., extended-spectrum β-lactamase-producing Enterobacteriaceae, vancomycin-resistant Enterococcus faecium, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus, have been identified by the Antimicrobial Availability Task Force of the Infectious Diseases Society of America as pathogens for which there is an immediate need for new drug development.
Methicillin-Resistant Staphylococcus Aureus (MRSA)
Staphylococcus aureus is a ubiquitous gram-positive bacterium that causes severe morbidity and mortality worldwide. S. aureus rapidly develops resistance to antibiotics. In the 1960s, the methicillin group of antibiotics (including cloxacillin) was introduced allowing the control of infections caused by S. aureus. However, in 1961, resistance to methicillin soon evolved by hospital-acquired methicillin resistant Staphylococcus aureus (HA-MRSA). Resistance to methicillin occurred due to the acquisition of a MecA gene carried on a mobile genetic element (Staphylococcal Cassette Chromosome mec; SCCmec).
Although, MRSA was mostly confined to hospital-acquired infections for a long period of time, in 1993, a new form of MRSA known as community-acquired MRSA (CA-MRSA) appeared in Western Australia. The incidence of community-associated infections was thought to emerge due to the development of hypervirulent and/or highly transmissible MRSA strains. CA-MRSA is currently a global health problem and is epidemic in the U.S.
In 2004, MRSA was reported as the most frequent cause of infection that was presented to emergency departments in the US. In 2005, in Egypt, it was reported that the percentage of MRSA from S. aureus isolates was 63% (n=243). CA-MRSA is mostly transmitted via skin-to-skin contact. Predisposing risk factors to CA-MRSA infection include skin trauma, injection drug use, and poor personal hygiene. Risk groups for CA-MRSA infection include professional athletes (contact sports), military personnel, children, and incarcerated individuals. Disease severity ranges from minor skin and soft tissue infections to severe life threatening complications; including fatal sepsis, necrotizing fasciitis and pneumonia. CA-MRSA is treated using oral antimicrobial agents including cotrimoxazole, clinamycin, tetracyclines (doxycycline and minocycline), rifampicin and fusidic acid. It is important to note that no clinically approved vaccine for the prevention of S. aureus infections is available.
The DNA genome of S. aureus is 2.8-2.9 Mb in size; composed of core and accessory genes. Most of the core genes are associated with metabolism and other housekeeping functions. However, some core genes were found not be linked to growth/survival but instead associated with S. aureus-specific virulence genes. Accessory genome usually consists of mobile genetic elements (MGEs) including bacteriophages, pathogenicity islands, chromosomal cassettes, genomic islands, and transposons. These are mostly responsible for virulence, drug and metal resistance, substrate utilization and miscellaneous metabolism. S. aureus isolates also often carry one or more free or integrated plasmids. These plasmids carry genes responsible for resistance to antibiotics, heavy metals, or antiseptics. Two main differences exist between CA-MRSA and HA-MRSA. The first is the presence of SCCmec (mobile genetic elements) types IV & V in CA-MRSA while HA-MRSA mainly harbor SCCmec types I, II, and III. Panton-Valentine leukocidin (PVL) exotoxin is found in severe skin infections and necrotizing pneumonia associated with CA-MRSA.
Viruses
Hepatitis B Virus (HBV)
Hepatitis B virus (HBV) currently infects about two billion patients worldwide leading to 600,000 deaths yearly. Disease chronicity rates differ according to the age group, where it is ≤5% in generally healthy infected adults and 80-90% in perinatally infected children. About 350 million patients suffer from chronic HBV worldwide. In 15-40% of Chronic HBV patients, cirrhosis with or without HCC develops. HBV accounts for 30% of cirrhotic and 50% of HCC cases, worldwide. In Egypt, the cumulative seroprevalence of HBV infection was reported to be 1.3% as determined by measuring hepatitis B surface antigen (HBsAg) using ELISA.
HBV can be transmitted through perinatal (vertical transmission), percutaneous or sexual routes. In countries with high seroprevalence of HBV (more than 8%), the disease is mostly acquired during childhood through perinatal or horizontal transmission. In countries with an intermediate seroprevalence (2-7%), the disease may be acquired during childhood or even at a later age (through sexual transmission, drug abuse or unsafe health practices). In low prevalence countries (seroprevalence less than 2%), the disease is usually acquired through sexual transmission or drug abuse. The U.S. Food and Drug Administration (FDA) has approved six drugs for HBV treatment, namely interferon-alpha, PEGylated IFN-a, lamivudine (cytidine analog), adefovir dipivoxil (dATP analog that function as chain terminator), entecavir (20-deoxyguanosine analog), and telbivudine (dTTP analog).
The HBV viral genome is a 3.2 kb circular partially duplex DNA molecule with the circularity maintained by 5′ cohesive ends. The negative DNA strand is responsible for mRNA transcription. The genome is composed of condensed coding regions having four overlapping open reading frames (ORFs). ORF P codes for a terminal protein on the minus strand as well as viral polymerase, ORF C codes for nucleocapsid structural protein as well as HBV e antigen (HBeAg), ORF S/pre-S codes for viral surface glycoproteins and ORF X codes for a transcriptional transactivator that is involved in HCC development. Mis-incorporations of nucleotides occur during viral replication due to lack of proofreading activity of DNA- and RNA-dependent DNA polymerase. This leads to the emergence of different HBV genotypes and subgenotypes. At least eight different genotypes (A-H) have been identified so far.
Human Immunodeficiency Virus (HIV)
Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS) and infects more than 33.3 million individuals worldwide, with 2.6 million new infections in 2009. HIV is regarded as a global epidemic and strikes the resource-poor regions of sub-Saharan Africa. In 2009, about 1.8 million) lives were lost due to AIDS of which 72% were in sub-Saharan Africa.
HIV infection causes destruction of CD4 T cells, and as the virus continues to replicate, it continues to cause a state of immune activation which overwhelms the homeostasis of the patient's immune system. This gravely compromises the body's immunity, and AIDS typically develops within 8-10 years. In the course of infection, various opportunistic infections take over such as those caused by M. tuberculosis, S. pneumonia, esophageal Candidiasis, and cytomegalovirus. This can occur as early as within one month of infection. HIV is transmitted through contact with infected bodily fluid such as blood, semen, and breast milk, and the infection can spread via syringe sharing, blood transfusion, sexual intercourse, and from pregnant mothers to their babies. Interestingly, it is estimated that 20-80% of HIV-infected individuals are unaware of their infection and would go on about their life with no protective measures for controlling infection spread.
The HIV virus is a spherical lentivirus belonging to the retroviruses family and ranges in diameter between 100 and 120 nm. It is composed of a lipid bilayer envelope enclosing a nucleocapsid which contains the viral genome; two copies of a 9.2 kb single-stranded positive-sense RNA molecule. Other proteins, e.g., integrase, a viral protease, and reverse transcriptase are also enclosed within the nucleocapsid including those vital for virus replication. There are two HIV types; HIV-1 and HIV-2, but the geographical spread of HIV-2 is rather limited. There is no vaccine for HIV. Costly and complex highly active antiretroviral therapy (HAART) is the currently available treatment. Early diagnosis of AIDS is an important factor in determining HAART efficacy.
Influenza A (H1N1 and H5N1)
Influenza is an infectious disease that is caused by the influenza viruses; RNA viruses of family Orthomyxoviridae. Influenza viruses infect both birds and mammals. Influenza is an air born infection transmitted through droplets or by direct contact with birds or animal droppings. Through the past few years major pandemic outbreaks of two influenza A viruses caused the world millions of deaths and loss of billions of dollars. These influenza A viruses were the swine flu (H1N1) influenza A virus and the avian flu (H5N1) influenza A virus.
The 2009 (H1N1) influenza pandemic more commonly, albeit inaccurately, known as the swine flu pandemic, bore a heavy global economic and societal burden. The pandemic infected about 50 million people, claimed over 18,449 lives, and spread to more than 214 countries between April 2009 and August 2010.
The influenza A (H5N1) virus known as avian flu, is an epizootic disease that infects both man and birds. There have been several outbreaks caused by H5N1 influenza A virus through the past years in Asia, Europe, the Near East, and Africa. Incidence is not expected to decrease in the next years.
The symptoms of H1N1 and H5N1 infections are similar to the seasonal influenza, e.g., fever, cough, sore throat, and myalgia. However, children, young adults, and individuals with cardiac or pulmonary problems, as well as pregnant women are quite susceptible to complications including pneumonia, encephalopathy, and secondary bacterial infections, which were among the causes of fatalities.
Subtypes H1N1 (swine flu) or H5N1 (avian flu), each of which are causative agents for pandemic influenza, belongs to the Orthomyxoviridae family. The virus consists of a negative-sense single stranded RNA genome enclosed within a lipid envelope. The influenza A virus 13.5 kb genome consists of 8 segments encoding 11 viral proteins. These proteins are: PA, PB1, and PB2 which are involved in the RNA-dependent RNA polymerase complex; nucleoprotein (NP); nonstructural proteins (NS1 and NEP), matrix proteins (M1 and M2), and the glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA and NA are the antigenic determinants upon which the subtypes of influenza A are classified. This airborne virus has a typical incubation period ranging from 1.5 to 3 days, but can reach up to 7 days, and spreads from human to human. It can be transmitted via exposure to aerosol droplets, e.g., through sneezing, or through contact with secretion containing the pathogen. The name “swine flu” is misleading, as the H1N1 virus that caused the 2009 pandemic is not the same as the one endemic in pigs, but is actually a new virus. The new virus was termed 2009 H1N1 influenza for clear discrimination. There was no evidence of pig to human transmission of the 2009 virus and the swift spread of the infection was due to human to human transmission. The avian flu (H5N1) is transmitted from birds to humans with little evidences of human to human transmission.
West Nile Virus (WNV)
WNV is a mosquito-transmitted virus, mainly by the species Culex, and infects different species including birds, horses, and humans. Humans and horses are considered dead-end hosts for the virus, but it is maintained in nature via bird-mosquito transmission. WNV cannot be transmitted between humans via casual contacts but can be through contaminated blood products, and vertically from mother to offspring intrauterinely or via breastfeeding. There is no vaccine currently available for humans against WNV. The virus was discovered in Uganda in 1937, but spread to United States and Canada in the late 1990s. Its epidemiology is variable, but is has subsequently become a considerable threat particularly in transfusion centers.
The incubation period of WNV ranges from 2 to 15 days and most infection—about 80% of cases—are asymptomatic. In symptomatic patients, the disease manifests usually as a mild self-limiting febrile condition, which can be associated with nausea, myalgia, headache, chills, and vomiting. However, about 5% of symptomatic patients develop neurological manifestations of the disease including encephalitis, meningitis, and acute flaccid paralysis. Individuals most susceptible to WNV infection and complications are those with compromised immunity such as those infected with human immunodeficiency virus, as well as older adults.
WNV belongs to the family Flaviviridae, the same family which encompasses hepatitis C virus, Japanese encephalitis viruses, and human immunodeficiency virus. It is a 50 nm enveloped positive strand RNA virus with a genome of about 11 kb. The genome is contained in an icosahedral nucleocapsid within a lipid envelope. The genome has a single open reading frame, flanked by untranslated regions on both terminals, and encodes a single polyprotein which is then cleaved by viral and cellular proteases to yield 10 proteins—3 structural proteins and 7 non-structural proteins.
Current Diagnostic Strategies
Acinetobacter
Current protocols for isolation and identification of Acinetobacter include the isolation on selective differential media (such as Herellea agar or Chromagar), biochemical testing and molecular approaches. Molecular diagnostic approaches are considered more reliable tests for genus and species identification than conventional methods. Detection of Acinetobacter resistant strains is achieved by agar diffusion methods and molecular assays. Agar diffusion methods involve the determination of the susceptibility pattern of the isolated strains by standard agar diffusion approach using sets of antibiotic discs representing different antibiotic classes. Advanced molecular methods such as PCR and Real-Time PCR are also used for detection of specific genes associated with Acinetobacter resistance.
Mycobacterium tuberculosis
The main strategies for TB diagnosis have not changed much for decades, and the primary detection methods of active infection rely on finding the TB bacilli in patient sputum smears, an approach that misses about 30-35% of positive cases and the detection rates are highly variable ranging from 20-80%. Detection rates below 20% are observed in HIV patients. Smear microscopy remains the primary identification tool especially in the developing countries. However, its accuracy depends on the bacterial load and the quality of the sputum specimen and the training of the laboratory technicians. Isolation and culturing of Mycobacterium on liquid or solid media is more sensitive method and allows for testing antibacterial sensitivity. However, culturing Mycobacterium requires biosafety facilities that are expensive to set up and maintain and require highly trained laboratory technicians. Some developing countries do not have a TB culturing facility at all, while in other countries TB culture is performed in national reference laboratories or in hospitals in large cities. Only few developing countries have the access to high quality sensitivity testing of first-line drugs and even fewer for testing second line drugs. Even when capacity exists TB diagnosis by culture still can take weeks because of the slow growth rate of mycobacteria. In most countries, TB culturing takes place in central laboratories. Therefore, clinical specimens often have to be sent to distant laboratories increasing processing time thus affecting the results.
Molecular detection lines based on PCR, Real-time PCR and microarray are used for the identification of mycobacterium and the detection of resistant strains Molecular methods might prove advantages regarding sensitivity and processing time, however performing these methods need highly equipped laboratories with highly trained staff. This will limit the benefits of the low income countries which represent the majority of HBCs which in turn will limit the impact of these new methods on the global TB control efforts.
The current facts regarding TB incidence and prevalence, together with socioeconomic status of the high burden countries, raise the need of developing new diagnostic tools. Optimum new diagnostic tools should be highly specific, highly sensitive, require low cost/low tech laboratory and minimal skilled labor, thus it can be widely used in low income countries and positively impact the global TB control efforts.
Nontuberculous Mycobacteria
Diagnosis of NTM requires clinical suspicion and exclusion of TB and lung malignancy in case of pulmonary manifestation as well chest radiograph. Laboratory tests available for diagnosis of NTM include culturing and smear microscopy, biochemical tests such as nitrate reduction, as well as molecular methods such as PCR and loop-mediated isothermal amplification (LAMP). Traditional culturing and phenotypic examination methods, albeit being cheap, are slow, tedious, and are of limited reproducibility and sensitivity. Additionally, M. Leprae cannot be cultured and the laboratory diagnosis of leprosy is based on histological examination of skin biopsies. PCR-based molecular assays are available gaining popularity due to their specificity and rapid turnaround time but their use is hindered by cost in developing countries.
Methicillin-Resistant Staphylococcus Aureus (MRSA)
Diagnostic assays for MRSA detection can be divided into culture-based methods as well as molecular assays. Culture-based methods can be further divided into conventional and rapid-culture based methods. Conventional culture-based methods depend on selective culturing in liquid and/or on solid media. These methods are time-consuming where the result is given to the patient after about 2-3 days, resulting in the development of severe complications as well as significant disease spread. Also it may give false-positive or false-negative results with sensitivity and specificity of 78-80% and 99%, respectively. More rapid culture-based methods utilizing chromogenic agars have been developed that produce results within 1.35-2.31 days. These culture agars incorporate a colorless chromogenic substrate that mimics a metabolic substrate. When the colorless chromogenic substrate is cleaved by a specific target bacterial enzyme, it becomes insoluble and colored. When the cleaved chromogen accumulates within the bacterial cell, the color builds up and the colony possessing the enzyme can be easily differentiated.
Currently available chromogenic media for MRSA diagnosis include ChromlD (bioMérieux, Marcy l'Etoile, France), CHROMagar MRSA (CHROMagar Microbiology, France; BD Diagnostics, Belgium), MRSA Select (BioRad Laboratories, Belgium), Chromogenic MRSA/Denim Blue agar (Oxoid, Basingstoke, UK), MRSA Ident agar (Heipha Gmbh, Eppelheim, Germany), Chromogen oxacillin S. aureus medium (Axon Labs AG, Stuttgart, Germany), and Oxacillin resistance screening agar base (ORSAB, Oxoid). Although chromogenic media are about 2 to 13 times more expensive than conventional media, it spares a number of subcultures, additional tests/reagents and technologist's time that are needed to confirm diagnosis in case of conventional culture-based MRSA detection. Sensitivity and specificity differ according to the media used, ranging from 40-100% and 44-100%, respectively.
A culture-based assay for MRSA detection has been developed (Baclite Rapid MRSA test; 3M Healthcare, Berkshire, UK). This assay detects ciprofloxacin-resistant MRSA strains by the measurement of adenylate kinase (AK) activity using bioluminescence. The total assay time is 5 hrs with assay sensitivity and specificity of 90.4% and 95.7%, respectively. However, the material cost of the assay is $9.5-12/test, which is higher than conventional culture-based methods. Another drawback is that cases of either CA-MRSA or HA-MRSA that are not resistant to ciprofloxacin will be missed.
Molecular methods provide many advantages over culture methods including higher sensitivity (lower detection limits), higher-throughput screening and faster detection (as low as 75 min), thus reducing risk of disease spread and progression. Available PCR methods for MRSA include the IDI-MRSA (GenOhm, San Diego, Calif.; BD Diagnostics), GeneXpert MRSA assay (Cepheid, Sunnyvale, Calif.), the GenoType MRSA Direct (Hain Lifescience, Nehren, Germany), the Hyplex StaphyloResist® PCR (BAG, Lich, Germany) and Lightcycler Staphylococcus and MRSA detection kit (LC assay, Roche Diagnostics, Mannheim, Germany). Sensitivity of PCR was found to be superior to cultural methods where a sensitivity of 93% has been reported. Specificity reported for PCR was 96%. However, the major drawback in molecular methods is their cost. For example, IDI-MRSA is a FDA cleared kit for the direct detection of MRSA from nasal specimens with high sensitivity and specificity. However, it is significantly more expensive than culture-based detection methods ($36.70/test).
Thus novel molecular methods that are cost-effective are highly needed to allow for the inexpensive as well as rapid detection of MRSA allowing control over disease spread and progression. The developed gold nanoparticle (AuNP)-based assay benefits from the unique physical properties of the AuNPs allowing for the sensitive, rapid and inexpensive detection of such deadly bacteria. It is important to note that the assay of the present invention uses unmodified AuNPs which makes the assay much simpler compared to other published assays utilizing probe-modified AuNPs.
Hepatitis B Virus (HBV)
Many markers are available for HBV diagnosis. Alanine aminotransferases (ALT) are significantly elevated in case of HBV acute infection and declines when viremia is cleared. In case of inactive carriers, ALT levels decline and normalize. Regarding anti-HBV antibodies, after exposure, anti-HBc IgM infection rises then declines, while, anti-HBc IgG rises and persists even after resolving of acute infection. In case of inactive carriers, anti-HBc IgM levels decline and normalize. Other markers for HBV detection include HBV antigens. HBsAg is a marker used for confirmation of acute infection that can be detected using enzyme immunoassays (EIA) around 6 weeks after exposure. If HBsAg persists for more than 6 months, this indicates chronic infection. HBeAg is a marker that indicates active viral replication. Recently, there is interest in the development of immunoassays that quantify (not only determine presence) HBeAg and HBsAg levels in patient blood. These assays may be used for therapeutic monitoring.
The final marker for infection is HBV DNA that is detected using molecular diagnostic assays. The presence of HBV DNA is an indication of active replication and can be detected at less than 6 weeks after exposure. In case of inactive carrier state, HBV DNA levels drop to less than 105 copies/mL.
Four main types of molecular assays have been developed for the diagnosis and management of HBV; namely quantitative viral load, genotyping, drug resistance mutation, and core promoter/precore mutation assays. Detection and quantitation of HBV DNA in plasma and serum has many advantages including; early detection of infection, allows for drug monitoring; helps assess disease activity in chronic patients, gold standard for the determination of HBV viral replication, confirms spontaneous remission or co-infection and finally detects occult infections (detectable HBV DNA in the absence of HBsAg). However, these tests are not well standardized, cut-off levels for inactive disease are still unclear and assays are relatively expensive.
Available viral load detection assays include signal amplification as well as target amplification assays. Signal amplification assays are less sensitive than target amplification assays, however, they are less prone to contamination. Examples of signal amplification assays include Digene Hybrid Capture assay (Digene Diagnostics; Corporation, Gaithersburg, Md.) and VERSANT HBV DNA 3.0 Assay (bDNA). Digene Hybrid Capture assay depends on the hybridization between HBV DNA and an RNA probe followed by their capture to an immobilized anti-RNA:DNA antibody. A chemiluminesence signal proportional to the HBV DNA level is then achieved by the addition of enzyme-linked antibodies. The quantification range is 1.4×105-1.7×109 copies/mL. The second signal amplification method is bDNA (HBV DNA 3.0 Assay; Siemens Medical Solutions Diagnostics; Tarrytown, N.Y.). Signal amplification in this case depends on a series of sequential DNA hybridizations. The dynamic range of the assay is 2×103 to 1×108 copies/mL.
Regarding target amplification assays, one of the first commercial HBV DNA PCR assays developed was the AMPLICOR HBV MONITOR test (Roche Diagnostics; Basel, Switzerland). This was followed later by a semi-automated test known as COBAS AMPLICOR HBV MONITOR test (Roche Diagnostics; Basel, Switzerland). The lower and upper limits of detection were 2×102 and 2×105 copies/mL, respectively. Recently, Real-time PCR assays have been developed. These assays have a broader dynamic range, are less prone to contamination and are faster than conventional PCR assays.
Commercially available Real-time PCR assays include the COBAS TaqMan HBV test (Roche Molecular Diagnostics; Pleasonton, Calif.; quantitative range: 1.7×102 to 8.5×108 copies/ml), the Abbott Real-Time HBV assay (Abbott Laboratories; Abbott Park, Ill.; Taqman probe; quantitative range: 34 to 3.4×109 copies/ml), The Real Art kit for the Rotor-Gene m instrument (Corbett Research; Sydney, Australia; molecular beacons; quantitative range: ˜102 to 6×108 copies/ml). Although Real-time PCR assays are highly sensitive, rapid and less prone to contamination, they are relatively expensive.
Many HBV genotyping assays have been developed including sequencing, INNO-LiPA, restriction fragment polymorphism (RFLP), multiplex PCR, oligonucleotide microarray chips, reverse dot blot, restriction fragment mass polymorphism (RFMP), invader assay, and Real-time PCR. Most of these assays are time consuming, expensive and/or require facilities and resources only available in a developed infrastructure.
Direct sequencing can identify known as well as new resistance mutations; this method can't detect mutants present in low concentrations. Thus hybridization-based methods have been developed and were found to be more sensitive (detect mutants present in low concentrations) and less labor intensive. However, they can't detect new mutations and require individual probes for each mutation to be detected. The second generation hybridization-based assay (INNO-LiPA DR, version 2.0) can detect mutations in reverse transcriptase enzyme at codons 80, 173, 180, and 204 (linked to lamivudine resistance) and at codons 181 and 236 (linked to adefovir resistance). The concordance with direct sequencing was ˜95%. However, this assay is still expensive and results are sometimes non-conclusive (faint bands).
Assays that detect Core Promoter/Precore Mutations that are linked to antiviral resistance include Affigene HBV Mutant VL19 [Sangtec Molecular Diagnostics AB] (hybridization/direct sequencing) and INNO-LiPA HBV PreCore (PCR plus hybridization). The INNO-LiPA HBV PreCore assay detects three mutations; namely basal core promoter nucleotides (1762 and 1764) and precore codon 28. INNO-LiPA HBV PreCore assays has high concordance with direct sequencing (˜90%) and is more efficient than direct sequencing in detecting mixed populations.
Novel molecular methods that are cost-effective are highly needed to allow for the inexpensive as well as rapid detection of active infection by detecting HBV DNA in serum. Also inexpensive and rapid assays are still needed for HBV genotyping and the detection of mutations linked to antiviral resistance. The developed AuNP-based assay benefits from the unique physical properties of AuNPs allowing for the sensitive, rapid and inexpensive detection of HBV DNA in serum, HBV genotyping and the detection of mutations linked to antiviral resistance. It is important to note that our assay uses unmodified AuNPs which makes the assay much simpler compared to other published assays utilizing probe-modified AuNPs.
Human Immunodeficiency Virus (HIV)
A variety of serological and molecular assays are employed for HIV detection in clinical specimens. The primary screening method for HIV diagnosis is the detection of its specific antibodies in patient serum or plasma using enzyme immunoassays (EIAs). HIV specific antibodies can be detected typically 3-6 weeks post-infection and within 12 weeks of infection it is detected in about 99% of cases. Another important serological test is the detection of HIV p24 antigen (viral capsid protein) whose levels begin to rise within the first 3 weeks of infection, making it an earlier marker than antibodies. Fourth-generation immunoassays allow its simultaneous detection with HIV antibodies. A positive serology result requires performance of a confirmatory test, the most common of which is the costly and time consuming Western blotting. Line immunoassays can also be used for confirmation. Rapid tests for antibody detection in saliva are also available and their sensitivity and specificity exceed 99% (OraQuick® Advance Rapid HIV-1/2). EIAs typically cost 0.5-1 USD per test, while rapid tests cost 1-3 USD.
Molecular testing has gained favor in HIV diagnosis on account of its sensitivity and significant reduction of window period. The utilization of molecular testing for donated blood screening in the US decreased the window period to 12-15 days. However, the main utility of molecular testing is viral load quantitation, and has special utility in determination of HIV status of infants born to infected mothers. This is due to the fact that maternal antibodies may be detected in infants for up to 15 months. The main limitation of molecular testing is being quite expensive and demanding in terms of equipment infrastructure and personnel training. Reverse transcription PCR is the basic molecular technology for HIV detection, but different technologies have been developed and commercialized including Real-time PCR and branched DNA detection.
Influenza A (H1N1 and H5N1)
Initially there was no diagnostic test with suitable clinical performance to detect the novel H1N1 virus, which may have allowed the virus to spread for possibly months in the population undetected, prior to the outbreak. In order to establish a diagnosis of H1N1 infection for a patient presenting with flu-like symptoms, a positive result by reverse transcription polymerase chain reaction (RT-PCR) or viral culture is required by the Centers for Disease Control (CDC). Both tests are costly, time-consuming, where PCR takes several hours while the gold standard; the viral culture requires 2-7 days. Viral culture is highly labor intensive and requires downstream characterization of the cultured virus by hemagglutination inhibition (HI) and NA inhibition tests, or PCR. It also falls short of the desired 100% sensitivity. The R-Mix viral culture (Diagnostic Hybrids, Athens, Ohio) has a sensitivity of 88.9% and a specificity of 100%, while RT-PCR (Luminex RVP; Luminex, Austin, Tex.) demonstrates sensitivity of 97.8% and a specificity of 100%. Both PCR and viral culture require highly equipped laboratories and trained personnel. Real-time RT-PCR is becoming a favored method for molecular diagnosis of 2009 H1N1 influenza, where the CDC has published a recommended protocol for Real-time RT-PCR that utilizes TaqMan® probes. Identification of 2009 H1N1 and its differentiation from seasonal influenza A requires the targeting of two genomic regions of the virus; the matrix gene (to determine it is influenza A) and different regions of the HA gene (to differentiate the 2009 H1N1 subtype).
Rapid influenza antigen detection assays are available and yield results within 30 minutes and are specific to 2009 H1N1 virus. These are mainly enzymatic activity optical assays or direct fluorescent antibody tests. However, their sensitivities are quite low and variable (10-70%) and cannot distinguish influenza A subtypes. This makes them unsuitable for excluding H1N1 infection in patients with influenza-like symptoms. The WHO recommended detection of influenza A H5N1 virus using reverse transcriptase (RT)-PCR employing specific primers for H5 gene (HA gene). However the RT-PCR is a time consuming and prone to cross contamination especially in high-throughput laboratories during outbreaks. Recently new methods based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) has been reported.
West Nile Virus (WNV): Current Diagnostic Strategies
Challenges preventing effective detection of WNV include the very low levels of viremia in human blood and tissues at time of symptoms onset, and the potential cross reaction from other flaviviruses. The gold standard for WNV detection is Plaque Reduction Neutralization Test (PNRT), and serological tests are the primary methods used in clinical diagnosis of the infection. Detection of anti-WNV IgM using the IgM antibody-capture ELISA (MAC-ELISA) are recommended immunoassays along with indirect IgG ELISA. MAC-ELISA can allow detection of acute infection in human serum or CSF (sampling 8-45 days after infection), and there are 4 commercial ELISA kits IgM for WNV IgM detection approved by the FDA. More specific immunofluorescence assays were also developed for detection of anti-WNV IgM. The sensitivities of different ELISA based assays for anti-WNV IgG and IgM range from 90 to 95% while the specificities range from 96%-100%. An important issue is the fact that sera positive by MAC-ELISA require PNRT confirmation due to potential cross-reactivity with antibodies against other flaviviruses. Unfortunately, PNRT requires 6 days and biosafety level 3 facilities due to use of live viruses, which diminishes the advantages of rapidity and low cost gained by using ELISA. Nevertheless, detection of anti-WNV IgM in CSF is direct evidence of the infection. The turnaround around time of ELISA, albeit fast compared to PNRT, is still inconvenient, as it takes 2 days to yield results due to overnight incubation. The more complex epitope blocking ELISA is also sometimes used due to being species independent. Another issue with use of MAC-ELISAs is the fact that IgM can persist for in serum for about a year, which would prevent discrimination between early and late stage infections. Also, if the serum sampled too early neither specific IgM or IgG would have been generated against WNV, thus yielding false negative results. This would mean the need for the more complex and expensive nucleic acid testing for the virus.
Various molecular assays are available for detection of WNV RNA including conventional RT-PCR, real-time RT-PCR with different chemistries, loop-mediated isothermal amplification (LAMP), and nucleic acid sequence-based amplification (NASBA). For almost a decade real-time RT-PCR has been used for screening human samples for WNV, and there are currently two FDA-approved commercial assays; TaqScreen West Nile Virus Test (Roche Molecular Systems, Pleasanton, Calif., USA), and Procleix® West Nile Virus Assay (Gen-Probe Inc., San Diego, Calif./Chiron Corporation, Emeryville, Calif., USA). Real-time RT-PCR assays based on TaqMan chemistry can detect WNV down to 0.1 PFU of viral RNA. SYBR Green-based assays are also available but are less sensitive. A major issue with molecular tests, in addition to cost, is the fact the used primers and probes may fail to detect new strains with new mutations, and may detect just one of the two WNV lineages. This has warranted efforts for targeting regions conserved in both lineages. Despite the analytical advantages offered by molecular assays their cost and complexity are still a hindrance to their use in limited-resource settings. Also, the turnaround time and susceptibility to interference continue to loom over the more common immunological assays.
The inventors have discovered that an unmodified gold nanoparticle-based (AuNP-based) colorimetric method can be used to directly detect unamplified or amplified microbial nucleic acids extracted from clinical specimens and have invented a simple, rapid, and sensitive colorimetric assay that can sensitively detect different pathogens in clinical samples even under field conditions. The invention provides a highly sensitive and specific way to detect unamplified target nucleic acids from samples containing nucleic acids from Acinetobacter, Mycobacterium, Staphylococcus, Hepatitis B Virus, Human Immunodeficiency Virus, Influenza virus, or West Nile Virus. The invention provides a way to avoid the cost, delay and risks of conventional method that require polynucleotide labeling and modification of gold nanoparticles. Thus, providing a simple, rapid and inexpensive way to detect these pathogens compared to conventional methods.
One embodiment of the invention is a method for detecting a nucleic acid of a microorganism selected from the group consisting of Acinetobacteria, mycobacteria, staphylococci, hepatitis B viruses, human immunodeficiency viruses, influenza viruses, and West Nile viruses comprising: contacting a sample suspected of containing the nucleic acids of one of these pathogens with an oligotargeter that binds specifically to nucleic acids from the pathogen and with gold nanoparticles, detecting the aggregation of nanoparticles, and detecting Acinetobacteria, mycobacteria, staphylococci, hepatitis B viruses, human immunodeficiency viruses, influenza viruses, or West Nile viruses in the sample when the nanoparticles aggregate in comparison with a control or a negative sample not containing the pathogen where the nanoparticles do not aggregate.
In this method a nucleic acid, a sample to be assayed for the presence Acinetobacteria, mycobacteria, staphylococci, hepatitis B viruses, human immunodeficiency viruses, influenza viruses, or West Nile viruses is obtained. The nature of the sample will vary depending on the organism or pathogen being tested for. Examples of biological samples include blood, plasma, serum, CSF, urine, sputum, and mucosal secretions. Other biological samples suspected of containing nucleic acids from the microorganism of interest may also be used.
Nucleic acids in the sample may be tested directed in the method or may be further isolated or purified from the sample. In some embodiments, the nucleic acids in the sample are amplified using methods such as the polymerase chain reaction (PCR).
The sample is then contacted with an oligotargeter sequence. The oligotargeter sequence is selected to be a complement of a nucleic acid sequence of Acinetobacteria, mycobacteria, staphylococci, hepatitis B viruses, human immunodeficiency viruses, influenza viruses, or West Nile viruses. An oligotargeter will be of a length sufficient to recognize and bind the target nucleic acid in a sample. Preferably, an oligotargeter will contain about 15 to 40 contiguous nucleotides that correspond to a genomic or non-genomic nucleic acid sequence of a target nucleic acid from one of the pathogens named above. All intermediate values within this range are contemplated as well as longer or shorter contiguous nucleotide sequences that function as oligotargeters within this method.
In some embodiments the oligotargeter may be modified to improve its stability or other functional properties. For example, the oligotargeter may be made from a modified nucleic acid that contains inosine, a modified base, or that has a chemically-modified phosphate backbone.
In other embodiments, the oligotargeter may be tagged. For example, its 5′ end can be conjugated to a FAM dye, fluorescent dye, or fluorophore whose emission can be quenched by gold nanoparticles. In such an embodiment, the presence of the target nucleic acid from the pathogen is detected by the emission of fluorescence.
The sample is contacted with a selected oligotargeter polynucleotide sequence under conditions that permit it to anneal to nucleic acids present in the tested sample. Double-stranded portions of nucleic acids in the sample are denatured or unfolded, generally by heating, to form single strands. Contact conditions are standardized or controlled to permit recognition and annealing between the oligotargeter sequence and a single-stranded portion of nucleic acid from the pathogen in the sample. Appropriate selections of salt concentration, buffer/pH selection, oligotargeter concentration, target concentration, and denaturation and annealing temperatures are made to enable recognition and annealing of the oligotargeter sequence to target nucleic acids in the sample. The conditions above are preferably selected to optimize the ability of the oligotargeter to bind to target nucleic acid in the sample. This annealing (hybridization) takes place before the addition or simultaneously in the presence of gold nanoparticles (AuNPs) under conditions which do not affect the stability and dispersity of the colloidal gold nanoparticles.
After sufficient time has passed to permit annealing (hybridization) between the target nucleic acids in the sample and the oligotargeter, gold nanoparticles are introduced. Unmodified colloidal gold nanoparticles that have not been conjugated to antibodies or other protein or carbohydrate ligands are generally employed in this step. Gold nanoparticles are spheroidal or spherical in shape and generally range in size from 2 nm to 80 nm. Subranges and intermediate values falling within this range are also contemplated for the gold nanoparticles used in the invention, including subranges of 2-12 nm, 12 to 20 nm, 20-40 nm, and 40-80 nm.
The concentration of gold nanoparticles is selected to permit interaction between the gold nanoparticles and free oligotargeter sequences that have not annealed to target nucleic acids may be used. Similarly, a buffer that permits the interaction of gold nanoparticles and free oligotargeters, such as a citrate buffer is used for this step. Preferably, the concentrations of ingredients for both the oligotargeter/target annealing step and the interaction of unbound or free oligotargeter with the gold nanoparticles are selected to optimize sensitivity and specificity of the detection of the target nucleic acid in a particular kind of sample.
After a sufficient time has passed for free oligotargeters and gold nanoparticles to interact, the color of the sample, oligotargeter and gold nanoparticle mixture is determined. Samples where the gold nanoparticles have not aggregated remain red, while samples where the gold nanoparticles have aggregated turn blue. When a threshold amount of free oligotargeters remains available to bind to the gold nanoparticles, they prevent gold nanoparticle aggregation by creating a repulsive electrostatic charge between the gold nanoparticles that prevents their aggregation. However, when this threshold amount of free oligotargeters is reduced by the annealing (hybridization) of the oligotargeters to complementary target nucleic acid in the sample, then there are insufficient free oligotargeters present to prevent aggregation of the gold nanoparticles. Thus, a blue color indicates the presence of the target nucleic acid sequence that is complementary to the oligotargeter sequence and has bound to it, thus preventing the oligotargeter from effective binding to the nanoparticles and inhibiting their aggregation. A colorimetric determination may be made visually (by eye) or by qualitative or quantitative mechanical or electronic means. Positive or negative control samples may be used to help make a colorimetric determination.
As an optional preliminary step, any nucleic acids present in a fresh or stored sample may be extracted, enriched, or concentrated by a conventional chemical or biochemical method, for example, by using a commercial nucleic acid purification kit. A sample may be diluted, serially diluted or titrated prior to its use in this method. Thus, a target nucleic acid from Acinetobacter, mycobacteria, staphylococcus, hepatitis B virus, human immunodeficiency virus, influenza virus, or West Nile Virus can be purified or isolated from other components of a biological sample prior to its contact with the oligotargeter and gold nanoparticles.
In some embodiments of this method, no amplification will be performed on any nucleic acids in the test sample. In other embodiments, nucleic acids in a sample may be amplified using PCR and selected primers, such as primers that amplify a particular polynucleotide sequence or gene of interest in the pathogen.
A specific embodiment of the invention is a method for detecting the presence of Acinetobacter in a sample, for example, detecting Acinetobacter baumannii. Exemplary biological samples that may be collected for this embodiment include those from the skin, oral cavity or respiratory tract of a subject.
Oligotargeters that selectively detect Acinetobacter are described by SEQ ID NOS: 23-66 (16s region), 71-82 (ITS region), 105-126 (23s region), 131-132 (5s region), and 151-169 (ISAba1-OXA-23).
For embodiments of this method that amplify an Acinetobacteria target nucleic acid, a sense primer selected the group consisting of SEQ ID NOS: 1-11 (16s region), 67-68 (ITS region), 83-93 (23 s region), 127-128 (5s region), and 133-141 (ISAba1-OXA-23) and an antisense primer selected from the group consisting of SEQ ID NOS: 12-22 (16s region), 69-70 (ITS region), 94-104 (23s region), 129-130 (5s region), and 142-150 (ISAba1-OXA-23) can be used to amplify nucleic acids in the sample which can then be identified using an AuNP-based method using an oligotargeter that binds to the amplified nucleic acid, such as those described by SEQ ID NOS: 23-66 (16s region), 71-82 (ITS region), 105-126 (23s region), 131-132 (5s region), and 151-169 (ISAba1-OXA-23).
A mycobacteria oligotargeter may be selected to bind to a conserved mycobacterial nucleic acid sequence found in at least two of M. tuberculosis, M. africanum, M. bovis, M. canetti and M. microti. Alternatively, mycobacteria oligotargeters may selectively bind target nucleic acids in M. tuberculosis but not in M. africanum, M. bovis, M. canetti and M. microti; may selectively bind to target nucleic acids in M. leprae, M. marinum, or other tuberculous mycobacteria, but not in M. tuberculosis, M. africanum, M. bovis, M. canetti and M. microti; or may selectively bind target nucleic acids of M. avium, M. kansasii, or another non-tuberculous mycobacterium, but not to nucleic acids of M. tuberculosis, M. africanum, M. bovis, M. canetti and M. microti.
A mycobacteria oligotargeter may be taken from a conserved region of the mycobacterial genomic sequences 16S (two regions), ITS region, IS6110 or the X-Conserved region.
Oligotargeters that selectively detect mycobacteria are described by SEQ ID NOS: 192-208 (16s region) and 215-225 (ITS region).
For embodiments of this method that amplify a target nucleic acid from mycobacteria, a sense primer selected the group consisting of SEQ ID NOS: 170-178 (16s region) and 209-210 (ITS region) and an antisense primer selected from the group consisting of SEQ ID NOS: 179-191 (16s region) and 211-214 (ITS region) can be used to amplify nucleic acids in the sample which can then be identified by an AuNP-based methods using at least one oligotargeter that binds to the amplified nucleic acid, such as those described by SEQ ID NOS: 192-208 (16s region) and 215-225 (ITS region).
A specific embodiment of the invention is a method for detecting the presence of Methicillin-Resistant Staphylococcus Aureus (MRSA) in a sample. Exemplary biological samples that may be collected for this embodiment include those from the anterior nares, throat, urinary tract, perineum, rectum, wounds, or sputum; or is obtained from a medical device.
Oligotargeters that selectively detect Methicillin Resistant Staphylococcus Aureus (MRSA) are described by SEQ ID NOS: 252-259 (16s region), 282-291 (23s region), 296-302 (ITS region), 319-332 (mecA), 345-352 (femA), 373-381 (gyrA), and 392-397 (spa).
For embodiments of this method that amplify a target nucleic acid from Methicillin-Resistant Staphylococcus Aureus (MRSA), a sense primer selected the group consisting of SEQ ID NOS: 238-244 (16s region), 260-270 (23s region), 292-293 (ITS region), 303-310 (mecA), 333-338 (femA), 353-362 (gyrA), and 382-386 (spa) and an antisense primer selected from the group consisting of SEQ ID NOS: 245-251 (16s region), 271-281 (23s region), 294 (ITS region), 311-318 (mecA), 339-344 (femA), 363-372 (gyrA), and 387-391 (spa) may be used to amplify nucleic acids in the sample which can then be detected by an AuNP-based method using at least one oligotargeter that recognizes the amplified nucleic acids.
A specific embodiment of the invention is a method for detecting the presence of HBV in a sample. Exemplary biological samples that may be collected for this embodiment include those from the blood, plasma or serum.
Oligotargeters that selectively detect HBV are described by SEQ ID NOS: 414 (target 1411-1880), 415-420 (CDC Polymerase [P]), 421-423 (HbsAg), 424-428 (RNA pre-alpha region), 429-430 (DNA enhancer 2), and 431-432 (CDS CO peptide).
For embodiments of this method that amplify a target nucleic acid from HBV, a sense primer selected the group consisting of SEQ ID NOS: 398 (target 1411-1880), 400 (HbsAg), 402 (RNA pre-alpha region), 404 (RNA pre-beta region), 406 (RNA epsilon element), 408 (CDS CO peptide), 410 (HbcAg) and 412 (CDC Polymerase [P]) and an antisense primer selected from the group consisting of SEQ ID NOS: 399 (target 1411-1880), 401 (HbsAg), 403 (RNA pre-alpha region), 405 (RNA pre-beta region), 407 (RNA epsilon element), and 409 (CDS CO peptide), 411 (HbcAg) and 413 (CDC Polymerase [P]) may be used to amplify nucleic acids in the sample which can then be detected by an AuNP-based method using at least one oligotargeter that recognizes the nucleic acid amplified by the sense and antisense primers, such as one selected from the group consisting of SEQ ID NOS: 414 (target 1411-1880), 415-420 (CDC Polymerase [P]), 421-423 (HbsAg), 424-428 (RNA pre-alpha region), 429-430 (DNA enhancer 2), and 431-432 (CDS CO peptide).
A specific embodiment of the invention is a method for detecting the presence of HIV-1 or HIV-2 in a sample. Exemplary biological samples that may be collected for this embodiment include those from the blood, plasma or serum.
Oligotargeters that selectively detect HIV-1 are described by SEQ ID NOS: 437-442 (gag) and those that identify HIV-2 by SEQ ID NOS: 447-454 (gag-pol).
For embodiments of this method that amplify a target nucleic acid from HIV-1 a sense primer selected the group consisting of SEQ ID NOS: 433 and 435 (gag) and an antisense primer selected from the group consisting of SEQ ID NOS: 434 and 436 (gag) may be used to amplify nucleic acid in the sample which may then be identified by an AuNP-based method using at least one oligotargeter that binds to the amplified nucleic acid, such as one described by SEQ ID NOS: 437-442 (gag).
For embodiments of this method that amplify a target nucleic acid from HIV-2 a sense primer selected the group consisting of SEQ ID NOS: 443 and 445 (gag-pol); and an antisense primer selected from the group consisting of SEQ ID NOS: 444 and 446 (gag-pol) may be used to amplify nucleic acid in the sample which may then be identified using an AuNP-based method using at least one oligotargeter that binds to the amplified nucleic acid, such as one selected from the group consisting of SEQ ID NOS: 447-454 (gag-pol).
A specific embodiment of the invention is a method for detecting the presence of Influenza in a sample, such as Influenza A (H1N1 & H5N1). Exemplary biological samples that may be collected for this embodiment include those from the respiratory tract.
Oligotargeters that selectively detect Influenza are described by SEQ ID NOS: 469-482 (HA gene), 493-506 (NA gene) and 513-524 (M1 gene).
For embodiments of this method that amplify a target nucleic acid from Influenza, a sense primer selected the group consisting of SEQ ID NOS: 455-461 (HA gene), 483-487 (NA gene) and 507-509 (M1 gene); and an antisense primer selected from the group consisting of SEQ ID NOS: 462-468 (HA gene), 488-492 (NA gene) and 510-512 (M1 gene) may be used to amplify nucleic acid in the sample. The amplified nucleic acid may be identified by an AuNP-based method using at least one oligotargeter that binds to the amplified nucleic acid, such as those described by SEQ ID NOS: 469-482 (HA gene), 493-506 (NA gene) and 513-524 (M1 gene).
A specific embodiment of the invention is a method for detecting the presence of West Nile Virus in a sample. Exemplary biological samples that may be collected for this embodiment include those from the blood, plasma, serum, or CSF. Oligotargeters that selectively detect West Nile virus are described by SEQ ID NOS: 539 (4603-5131 target), 550 (5′UTR), 551-553 (Mat_Peptide_2), 554-555 (Mat_Peptide_1), 556-557 (Mat_Peptide_3), 558-563 ((Mat_Peptide_5), 564-569 (Mat_Peptide_6), 570-571 (Mat_Peptide_7), 572-573 (Mat_Peptide_8), 574-581 (Mat_Peptide_9), 582-583 (Mat_Peptide_10), 584-587 (Mat_Peptide_12), 588-596 (Mat_Peptide_13) and 597-601 (3′UTR).
For embodiments of this method that amplify a target nucleic acid from West Nile virus using at least a pair of sense and antisense primers selected the group consisting of SEQ ID NOS: 525 (4603-5131 target), 527 (5′UTR), 529 (Mat_Peptide), 531 (Mat_Peptide), 533 (Mat_Peptide), 535 (Mat_Peptide), 537 (Mat_Peptide), 539 (Mat_Peptide), 541 (Mat_Peptide_9), 543 (Mat_Peptide_10), 545 (Mat_Peptide_12), and 547 (3′UTR) and an antisense primer selected from the group consisting of SEQ ID NOS: 526 (4603-5131 target), 528 (5′UTR), 530 (Mat_Peptide), 532 (Mat_Peptide), 534 (Mat_Peptide), 535 (Mat_Peptide), 536 (Mat_Peptide), 540 (Mat_Peptide_8), 542 (Mat_Peptide_9), 544 (Mat_Peptide_10), 546 (Mat_Peptide_12), and 548 (3′UTR), may be used to amplify nucleic acid in the sample which can then be detected by an AuNP-based method using at least one oligotargeter that binds to the nucleic acid amplified by the sense and antisense primers, such as those described by SEQ ID NOS: 539 (4603-5131 target), 550 (5′UTR), 551-553 (Mat_Peptide_2), 554-555 (Mat_Peptide_1), 556-557 (Mat_Peptide_3), 558-563 ((Mat_Peptide_5), 564-569 (Mat_Peptide_6), 570-571 (Mat_Peptide_7), 572-573 (Mat_Peptide_8), 574-581 (Mat_Peptide_9), 582-583 (Mat_Peptide_10), 584-587 (Mat_Peptide_12), 588-596 (Mat_Peptide_13) and 597-601 (3′UTR).
For the specific embodiments above, samples can be isolated or obtained from various kinds of patients or subjects. For example, they may be obtained from a subject under treatment for an infection or exposure to said microorganism; a sample may represent one or more longitudinal acquired samples from a subject undergoing treatment for an infection or exposure to said microorganism; or a sample can be obtained from a subject suspected of having an asymptomatic or latent infection by the microorganism.
The detection methods as disclosed herein may be practiced in a manner that helps determine the general or specific identity of one of the pathogens, such as its genus, species, sub-species, serotype, subtype, or strain. This may be accomplished by the selection of an oligotargeter or, for embodiments using amplification, a combination of primers and oligotargeters, that identifies unique genetic features of particular subtype of a pathogen or that identifies one or more single nucleotide polymorphisms in an amplified or unamplified nucleic acid of the microorganism. Similarly, one may determine the pathogenicity or drug resistance of a particular subtype or strain microorganism by contacting the sample with an oligotargeter that binds to a polynucleotide sequence of the microorganism that is associated with its pathogenicity or drug-resistance.
Another aspect of the invention pertains to kits for detecting Acinetobacter, mycobacteria, staphylococcus, hepatitis B virus, human immunodeficiency virus, influenza virus, or West Nile Virus. These embodiments include the following:
A kit for detecting Acinetobacteria comprising:
gold nanoparticles,
at least one oligotargeter that binds to nucleic acid of an Acinetobacterium, and optionally,
at least one: biological sample preservative or additive, buffer for extracting Acinetobacteria nucleic acid, modified silica nanoparticles, column or other device for purifying Acinetobacteria nucleic acids, reaction buffer, negative control sample, positive control sample, Acinetobacteria primer, Acinetobacteria probe, container, a colorimetric chart, packaging material, or instruction for use in detecting Acinetobacteria.
A kit that comprises at least one Acinetobacteria oligotargeter selected from the group consisting of SEQ ID NOS: 23-66 (16s region), 71-82 (ITS region), 105-126 (23s region), 131-132 (5s region), and 151-169 (ISAba1-OXA-23).
A kit that comprises a sense primer selected the group consisting of SEQ ID NOS: 1-11 (16s region), 67-68 (ITS region), 83-93 (23 s region), 127-128 (5s region), and 133-141 (ISAba1-OXA-23); an antisense primer selected from the group consisting of SEQ ID NOS: 12-22 (16s region), 69-70 (ITS region), 94-104 (23s region), 129-130 (5s region), and 142-150 (ISAba1-OXA-23); and at least one oligotargeter that binds to the nucleic acids amplified by the sense and antisense primers; this oligotargeter may be selected from the group consisting of SEQ ID NOS: 23-66 (16s region), 71-82 (ITS region), 105-126 (23s region), 131-132 (5s region), and 151-169 (ISAba1-OXA-23).
A kit for detecting mycobacteria comprising:
gold nanoparticles,
at least one oligotargeter that binds to nucleic acid of a mycobacterium, and optionally,
at least one: biological sample preservative or additive, buffer for extracting mycobacteria nucleic acid, modified silica nanoparticles, column or other device for purifying mycobacteria nucleic acids, reaction buffer, negative control sample, positive control sample, mycobacteria primer, mycobacteria probe, container, a colorimetric chart, packaging material, or instruction for use in detecting mycobacteria. A kit that comprises a mycobacteria oligotargeter selected from the group consisting of SEQ ID NOS: 192-208 (16s region) and 215-225 (ITS region).
A kit that comprises a sense primer for mycobacteria selected from the group consisting of SEQ ID NOS: 170-178 (16s region) and 209-210 (ITS region); an antisense primer for mycobacteria selected from the group consisting of SEQ ID NOS: 179-191 (16s region) and 211-214 (ITS region); and at least one oligotargeter that binds to the amplified nucleic acid which may be selected from the group consisting of SEQ ID NOS:192-208 (16s region) and 215-225 (ITS region).
A kit for detecting staphylococcus comprising:
gold nanoparticles,
at least one oligotargeter that binds to nucleic acid of a staphylococcus, such as MRSA, and optionally,
at least one: biological sample preservative or additive, buffer for extracting staphylococcus nucleic acid, modified silica nanoparticles, column or other device for purifying staphylococcus nucleic acids, reaction buffer, negative control sample, positive control sample, staphylococcus primer, staphylococcus probe, container, a colorimetric chart, packaging material, or instruction for use in detecting staphylococcus. A kit that comprises an staphylococcus oligotargeter selected from the group consisting of SEQ ID NOS: 252-259 (16s region), 282-291 (23s region), 296-302 (ITS region), 319-332 (mecA), 345-352 (femA), 373-381 (gyrA), and 392-397 (spa).
A kit that comprises a sense primer selected the group consisting of SEQ ID NOS: 238-244 (16s region), 260-270 (23s region), 292-293 (ITS region), 303-310 (mecA), 333-338 (femA), 353-362 (gyrA), and 382-386 (spa); an antisense primer selected from the group consisting of SEQ ID NOS: 245-251 (16s region), 271-281 (23s region), 294 (ITS region), 311-318 (mecA), 339-344 (femA), 363-372 (gyrA), and 387-391 (spa); and at least one oligotargeter that binds to the nucleic acids amplified by the sense and anti sense primers. This oligotargeter may be selected from the group consisting of SEQ ID NOS: 252-259 (16s region), 282-291 (23s region), 296-302 (ITS region), 319-332 (mecA), 345-352 (femA), 373-381 (gyrA), and 392-397 (spa).
A kit for detecting HBV (Hepatitis V virus) comprising:
gold nanoparticles,
at least one oligotargeter that binds to nucleic acid of HBV, and optionally,
at least one: biological sample preservative or additive, buffer for extracting HBV nucleic acid, modified silica nanoparticles, column or other device for purifying HBV nucleic acids, reaction buffer, negative control sample, positive control sample, HBV primer, HBV probe, container, a colorimetric chart, packaging material, or instruction for use in detecting HBV.
A kit that comprises at least one HBV oligotargeter selected from the group consisting of SEQ ID NOS: 414 (target 1411-1880), 415-420 (CDC Polymerase [P]), 421-423 (HbsAg), 424-428 (RNA pre-alpha region), 429-430 (DNA enhancer 2), and 431-432 (CDS CO peptide).
A kit that comprises a sense primer selected the group consisting of SEQ ID NOS: 398 (target 1411-1880), 400 (HbsAg), 402 (RNA pre-alpha region), 404 (RNA pre-beta region), 406 (RNA epsilon element), 408 (CDS CO peptide), 410 (HbcAg) and 412 (CDC Polymerase [P]); an antisense primer selected from the group consisting of SEQ ID NOS: 399 (target 1411-1880), 401 (HbsAg), 403 (RNA pre-alpha region), 405 (RNA pre-beta region), 407 (RNA epsilon element), and 409 (CDS CO peptide), 411 (HbcAg) and 413 (CDC Polymerase [P]); and at least one HBV oligotargeter that binds to the amplified nucleic acid. This oligotargeter may be selected from the group consisting of SEQ ID NOS: 414 (target 1411-1880), 415-420 (CDC Polymerase [P]), 421-423 (HbsAg), 424-428 (RNA pre-alpha region), 429-430 (DNA enhancer 2), and 431-432 (CDS CO peptide).
A kit for detecting HIV (Human Immunodeficiency virus) comprising:
gold nanoparticles,
at least one oligotargeter that binds to nucleic acid of HIV, and optionally,
at least one: biological sample preservative or additive, buffer for extracting HIV nucleic acid, modified silica nanoparticles, column or other device for purifying HIV nucleic acids, reaction buffer, negative control sample, positive control sample, HIV primer, HIV probe, container, a colorimetric chart, packaging material, or instruction for use in detecting HIV.
A kit that comprises at least one HIV-1 oligotargeter selected from the group consisting of SEQ ID NOS: 437-442 (gag).
A kit that comprises at least one HIV-2 oligotargeter selected from the group consisting of SEQ ID NOS: 447-454 (gag-pol).
A kit that comprises a sense primer for HIV-1 selected the group consisting of SEQ ID NOS: 433 and 435 (gag); an antisense primer for HIV-1 selected from the group consisting of SEQ ID NOS: 434 and 436 (gag); and at least one HIV-1 oligotargeter that binds to the amplified nucleic acid. This oligotargeter may be selected from the group consisting of SEQ ID NOS: 437-442 (gag).
A kit that comprises a sense primer for HIV-2 selected the group consisting of SEQ ID NOS: 443 and 445 (gag-pol); an antisense primer for HIV-2 selected from the group consisting of SEQ ID NOS: 444 and 446; and at least one HIV-2 oligotargeter that binds to nucleic acid amplified by the sense and antisense primers. This HIV-2 oligotargeter may be selected from the group consisting of SEQ ID NOS: 447-454 (gag-pol).
A kit for detecting Influenza virus, such as Influenza A (H1N1) comprising:
gold nanoparticles,
at least one oligotargeter that binds to nucleic acid of Influenza virus, and optionally, at least one: biological sample preservative or additive, buffer for extracting Influenza virus nucleic acid, modified silica nanoparticles, column or other device for purifying Influenza virus nucleic acids, reaction buffer, negative control sample, positive control sample, Influenza virus primer, Influenza virus probe, container, a colorimetric chart, packaging material, or instruction for use in detecting Influenza virus.
A kit that comprises at least one Influenza virus oligotargeter selected from the group consisting of SEQ ID NOS: 469-482 (HA gene), 493-506 (NA gene) and 513-524 (M1 gene).
A kit that comprises a sense primer selected the group consisting of SEQ ID NOS: 455-461 (HA gene), 483-487 (NA gene) and 507-509 (M1 gene); an antisense primer selected from the group consisting of SEQ ID NOS: 462-468 (HA gene), 488-492 (NA gene) and 510-512 (M1 gene), and at least one Influenza virus oligotargeter that binds to the nucleic acid amplified by the sense and antisense primers, such as one selected from the group consisting of SEQ ID NOS: 469-482 (HA gene), 493-506 (NA gene) and 513-524 (M1 gene).
A kit for detecting West Nile virus comprising:
gold nanoparticles,
at least one oligotargeter that binds to nucleic acid of West Nile virus, and optionally, at least one: biological sample preservative or additive, buffer for extracting West Nile virus nucleic acid, modified silica nanoparticles, column or other device for purifying West Nile virus nucleic acids, reaction buffer, negative control sample, positive control sample, West Nile virus primer, West Nile virus probe, container, a colorimetric chart, packaging material, or instruction for use in detecting West Nile virus.
A kit that comprises a West Nile virus oligotargeter selected from the group consisting of SEQ ID NOS: 539 (4603-5131 target), 550 (5′UTR), 551-553 (Mat_Peptide_2), 554-555 (Mat_Peptide_1), 556-557 (Mat_Peptide_3), 558-563 ((Mat_Peptide_5), 564-569 (Mat_Peptide_6), 570-571 (Mat_Peptide_7), 572-573 (Mat_Peptide_8), 574-581 (Mat_Peptide_9), 582-583 (Mat_Peptide_10), 584-587 (Mat_Peptide_12), 588-596 (Mat_Peptide_13) and 597-601 (3′UTR).
A kit that comprises a sense primer for West Nile virus selected from the group consisting of SEQ ID NOS: 525 (4603-5131 target), 527 (5′UTR), 529 (Mat_Peptide), 531 (Mat_Peptide), 533 (Mat_Peptide), 535 (Mat_Peptide), 537 (Mat_Peptide), 539 (Mat_Peptide), 541 (Mat_Peptide_9), 543 (Mat_Peptide_10), 545 (Mat_Peptide_12), and 547 (3′UTR); and an antisense primer selected from the group consisting of SEQ ID NOS: 526 (4603-5131 target), 528 (5′UTR), 530 (Mat_Peptide), 532 (Mat_Peptide), 534 (Mat_Peptide), 535 (Mat_Peptide), 536 (Mat_Peptide), 540 (Mat_Peptide_8), 542 (Mat_Peptide_9), 544 (Mat_Peptide_10), 546 (Mat_Peptide_12), and 548 (3′UTR); and one or more West Nile virus oligotargeters that bind to the nucleic acid amplified by the sense and antisense primers, such as one selected from the group consisting of SEQ ID NOS: 539 (4603-5131 target), 550 (5′UTR), 551-553 (Mat_Peptide_2), 554-555 (Mat_Peptide_1), 556-557 (Mat_Peptide_3), 558-563 ((Mat_Peptide_5), 564-569 (Mat_Peptide_6), 570-571 (Mat_Peptide_7), 572-573 (Mat_Peptide_8), 574-581 (Mat_Peptide_9), 582-583 (Mat_Peptide_10), 584-587 (Mat_Peptide_12), 588-596 (Mat_Peptide_13) and 597-601 (3′UTR).
A colorimetric assay has been developed using unmodified AuNPs for the direct detection of unamplified DNA or RNA in biological fluids containing different kinds of microbes. In some embodiments the DNA or RNA may be amplified and it others it is not necessary. The assay has a detection limit of 0.6-0.8 ng (0.0028-0.00373 pM/uL) in samples including sputum, pleural fluid, blood, cerebrospinal fluid, tissue biopsy samples, feces, bacterial isolates, and pus from a wound. The highly sensitive assay of the invention can be applied for tracking microbial titers. Method sensitivity can be controlled by changing buffers and or the kind or concentration of oligotargeter as well as the concentration of gold nanoparticles. The lower the concentration of the probes used the higher the sensitivity. It is logical that using oligotargeters to target repeats should increase the sensitivity but might affect the specificity on strain level. However, oligotargeters have been identified that target a repeat region in microbes that, according to BLAST similarity check, would be highly specific only to particular microbes.
The invention has several other advantages in addition to its high sensitivity including excellent specificity, short turnaround time, and cost effectiveness. The assay is economical, for example, 1 gram of gold is sufficient to prepare 1 liter of 15 nm gold nanoparticles and only about 10 μL of gold nanoparticles are required per assay. Thus, based on a cost of 1 gram of gold chloride of about 200 euros, the assay is highly cost effective. While the cost of DNA or RNA extract kits used to extract microbial nucleic acid from clinical samples may vary, this cost generally ranges between 100-200 euros for 50 extractions and thus the overall cost of this assay is low, especially compared to more complicated prior art assays. Moreover, the use of AuNPs eliminates the need for expensive detection instrumentation and does not require functionalization of the AuNPs, the oligotargeter, or the target.
Moreover, this assay may be adapted into a quantitative test by spectrophotometric quantification of the resulting blue color against a standard curve or developing a fluorometric version of the test by utilization of the size and distance nanoparticle surface energy transfer (NSET) properties of AuNPs.
The assay of the invention may be further modified to detect SNPs of sequences of a microorganism, for example to discriminate between genotypes, variants or even quasispecies by manipulating the annealing temperature of the oligotargeters. This has great implications for microbiological genotyping, subtyping, and monitoring of factors correlated to treatment of a condition, disorder or disease.
The invention permits use of unmodified AuNPs for direct detection of unamplified microbe nucleic acids in clinical specimens and may be competitively used in place of other commercial immunoassays and RT-PCR methods as routine tests for management of patients infected with particular microorganisms.
The following abbreviations and terms appear herein.
AuNPs: Gold Nanoparticles. These are generally spheroidal or spherical and range in diameter from 2 to 80 nm. Unmodified AuNPs have not been bound to protein or carbohydrate ligands such as antibodies, lectins, or nucleic acids. The term “gold nanoparticle” refers to spherical gold nanoparticles. Generally, the gold nanoparticles are produced by citrate reduction method and have an average diameter ranging from 2.0 nm to 100 nm, preferably, an average diameter ranging from 10 to 25 nm, and more preferably from 15 to 20 nm. When the size of the gold nanoparticle is too small, then performance is reduced because surface-plasmon resonance would be reduced and completely abolished for particles <2 nm and the color change will not be observed and when it is too large, then performance is reduced because the aggregation affinity of the nanoparticles would be higher leading to false positive results.
The gold nanoparticles used in the invention may be produced or synthesized by methods known in the art, such as those described above in the background section. Alternatively, exemplary methods include (a) by reduction of chloroauric acid with sodium borohydride; (b) By reduction of chloroauric acid with hydrogen peroxide; or (c) by a single phase microemulsion method. These methods of producing gold nanoparticles are hereby incorporated by reference to the articles cited above.
Sample describes a material suspected to contain a microorganism to be assayed for detection. Generally, a biological sample is obtained from a subject suspected of having been exposed to or having a microbial infection. Biological samples include whole blood, plasma or serum, or other bodily fluids that may contain the microorganism. These may include, for example, plasma, serum, spinal fluid, lymph fluid, secretions from the respiratory, gastrointestinal, or genitourinary systems including bronchial lavage, tears, saliva, milk, urine, semen, skin swabs, tissue biopsies, and red or white blood cells or platelets. Samples may also be obtained from tissue cell culture, such as cultured leukocytes, and constitute cells, including recombinant cells, or medium in which a microorganism may be detected. In some cases a tissue sample may be used in the assay or processed for use in the assay, for example, by a conventional method used to extract the microbial nucleic acids from the sample.
A sample may also be processed to remove non-nucleic acid components or to isolated or further purify nucleic acids it contains by a capture method that captures at least one biological material of interest. This method comprises contacting a sample suspected of containing the nucleic acid to be purified or isolated with a substrate containing iron oxide, gold, or silver nanoparticles, quantum dots or silica nanoparticles for a time and under conditions sufficient for the material to bind to these materials and optionally eluting the captured nucleic acid or other component from the substrate. For example, a sample suspected to contain Acinetobacter or mycobacteria nucleic acids is contacted with silica nanoparticles for a time and under conditions sufficient for the material to bind to the silica nanoparticles. The captured nucleic acid on the silica nanoparticles can then be washed or processed, and the eluted from the silica nanoparticles for further purification or characterization of the captured and released material. In this method, silica nanoparticles can be conjugated to a ligand that is a nucleic acid complementary to a nucleic acid to be detected or that is an aptamer that binds to the nucleic acid to be detected. For example, the silica nanoparticles can be conjugated to a ligand that binds to material containing or associated with the nucleic acid to be detected and the method can further involve isolating or purifying the nucleic acid from the material bound to the silica nanoparticles. Those of skill in the art may select various ligands for use in this capture method. Examples of such ligands include antibodies or fragments of antibodies containing binding sites such as those of IgA, IgD, IgE, IgG, IgM and the various subtypes of these kinds of antibodies. This capture method may be employ a ligand that is an aptamer that binds to a protein, carbohydrate, lipid or other material associated with the nucleic acid to be captured or detected. It also may employ ligands that bind directed to a material, such as DNA or RNA or a complex or aggregate of a nucleic acid with a peptide, polypeptide or protein, to be captured or detected. The ligand in this capture method may comprise various lectins, such as one or more mannose binding lectin(s), one or more N-acetyl glucosamine(s) with or without sialic acid binding lectin(s); one or more galactose/N-acetylgalactosamine binding lectin(s); one or more N-acetylneuraminic acid binding lectin(s); or one or more fucose binding lectin(s).
The ligand used in this method may be a specific probe for the nucleic acid to be detected that is conjugated to at least one member selected from the group consisting of iron oxide, gold, silver, quantum dots and silica nanoparticles of different sizes. The method may further involve extracting the nucleic acid from the material containing it or from the material from which it is associated which has been bound to said ligand. It also may comprise contacting the sample with nanoparticles selected from the group consisting of iron oxide, gold, silver, quantum dots and silica nanoparticles, which have been conjugated to a probe comprising an oligotargeter sequence. For example, the sample may be contacted with at least one oligotargeter as disclosed herein.
Alternatively, a material containing the nucleic acids, such as an aggregate containing nucleic acid, may be captured by the substrates described above. Similarly, a non-nucleic acid component of a biological sample or undesired nucleic acid can be separated and removed from a nucleic acid of interest in a biological sample may be captured thus removing it from the rest of the sample containing the nucleic acid of interest.
Isolated or purified microorganism describes one that has been removed from its original environment, such as from the skin, blood or respiratory system of a host. It also encompasses a biological sample that has been processed to remove some or all of the contaminants or substances associated with the microorganism. Contaminants include host components or components of other kinds of microorganisms, such as nucleic acids from the host other microorganisms in a biological sample. A microorganism may also be isolated or purified from tissue culture or from a microbial culture.
Preservative or additive for a sample includes additives such as heparin or EDTA. The term also includes other agents which prevent degradation of microbial DNA or RNA or permit microbial DNA or RNA to be easily recognized in the method of the invention. These include normal saline or commercially available preservatives such as the one found in PAX gene tubes. The term “extraction buffer” refers to agents and materials useful for extracting, purifying or isolating microbial DNA or RNA from a biological sample.
Denaturation refers to a process of unfolding microbial nucleic acids or separating the strands of a duplex nucleic acid. For example, a nucleic acid may be denatured by heating it to a temperature of 65, 75, 85, 90, and 95-100° C. Denaturation may also be facilitated by addition of other ingredients such as salts, formamide, or sodium hydroxide.
Hybridization buffer refers to a buffer that permits hybridization to occur between an oligotargeter sequence and a target nucleic acid, for example, 10 mM phosphate buffered saline (PBS), pH 7.0. Samples are admixed with the oligotargeter in hybridization buffer and subsequently denatured and annealed prior to admixture with gold nanoparticles. A preferred buffer is phosphate-buffered saline (“PBS”), pH 7.0-7.4. Monovalent cation (e.g., sodium or potassium) salt concentration can range from 50 mM to 300 mM. Suitable hybridization buffers and protocols are well-known in the art and are incorporated by reference to Maniatis, et al., Molecular Cloning: A Laboratory Manual, 2nd edition or Current Protocols in Molecular Biology, vol. 1 (updated October, 2010). Salt concentration is selected based on the volume and concentration of the gold nanoparticles.
Oligotargeter describes a polynucleotide that binds to a microbial nucleic acid. An oligotargeter forms binds via nucleic acid complementarity to a nucleic acid sequence in a target region of the microbial genomic nucleic acid. The oligotargeter will be long enough to bind to microbial nucleic acid in a sample. Preferably, it will comprise 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 bp. If the sequence is less than 19 bp, then performance will be reduced because shorter sequence will bind to non-specific sequences which would result in false positive results or if it exceeds 40 bp, then performance may be reduced because of dimerization and hairpin formation in the oligotargeter sequence risking an increase in false positive results. The oligotargeter may correspond to any portion of genomic RNA or DNA or its cellular nucleic acid components. However, it preferably will be selected to bind to a highly conserved portion and can be used to differentiate between different genera, species, subtypes, strains or variants of a microorganism. For detection of infection by a particular microorganism, preferably the oligotargeter will bind to a genomic sequence shared different pathogenic variants or subtypes of the microorganism causing human infection. For example, an oligotargeter may be selected to bind to a nucleic acid sequence conserved in the genomic DNA of mycobacteria.
Alternatively, an oligotargeter can be selected to distinguish among strains of an organism by making it complementary to subtype, species or strain-specific nucleic acids or designing it to bind to target nucleic acids containing one or more SNPs characteristic of a subtype of a pathogen. Genomic sequences can retrieved from GenBank or other nucleic acid databases and alignments of sequences from different subtypes of a pathogen performed using BLAST or other alignment software.
Modified oligotargeter is one that may contain one or more modified bases or contain a modification to or replacement of the phosphate backbone structure of a conventional oligonucleotide but otherwise substantially maintain its ability to hybridize to a target sequence, such as sequences derived from target genomic DNA or cellular RNA. For example, a modification to oligotargeter sequence that increases stability or resistance to degradation or improves binding specificity or sensitivity may be made. Examples of modifications to increase nuclease resistance of the oligotargeter include the following: (a) phosphorothioate modified sequence (where one of the oxygen on the phosphate of phosphodiester bond is replaced with a sulphur atom); (b) 3′-propryl group (C3 spacer, adding a propyl group at the 3′ end); and (c) Inverted end (3′-3′ linkage), though other modifications known to those in the art may also be employed.
For some applications an oligotargeter or modified oligotargeter may contain one, two, three, four or more degenerate bases, which can base pair with A, T, G, C and/or U. Degenerate bases may be incorporated into an oligotargeter to increase its affinity for the mycobacteria target sequence. For example, an oligotargeter containing one, two, three, four or more degenerate bases (e.g., inosine) in its oligonucleotide sequence can be used to overcome or compensate for a mutation that may occur within the same genotype and subtype (quasi species). Inosine resembles guanine, but without the 2-amino group, and can form stable (wobble) base pairs with adenine, cytosine and uracil that are similar in terms of interaction strength. Therefore, inosine in a probe can bind to perfectly complementary polynucleotide or ones that have mismatches at the location of the inosine to form duplex structures of comparable stability.
Variants of the oligotargeters disclosed herein having at least 80, 85, 90, 95% sequence identity or similarity or having one, two, three, four, five, six, seven, eight, nine or ten deletions, substitutions, insertions to the oligotargeter sequences described herein may be employed so long as they competently bind to the target sequence. For example, an oligotargeter variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 terminal nucleotides added to its 5′ and/or 3′ termini so long as these additions do not substantially affect its ability to bind to the target sequence.
With regard to the nucleic acid sequences described herein, the terms “percentage identity” and “percentage similarity” refer to a measure of the degree of identity or similarity of two sequences based upon an alignment of the sequences which maximizes identity or similarity between aligned nucleotides, and which is a function of the number of identical or similar nucleotides, the number of total nucleotides, and the presence and length of gaps in the sequence alignment. A variety of algorithms and computer programs are available for determining sequence similarity using standard parameters. Sequence similarity can be determined by the BLASTn program for nucleic acid sequences, which is available through the National Center for Biotechnology Information (http://_www.ncbi.nlm.nih.gov/blast/Blast.cgi?PAGE=Nucleotides)(last accessed Apr. 9, 2012). The percent identity of two nucleotide sequences may be made using the BLASTn preset “search for short and near exact matches” using a word size of 7 with the filter off, an expect value of 1,000 and match/mismatch of 2/−3, gap costs existence 5, extension 2; or standard nucleotide BLAST using a word size of 11, filter setting “on” (dust) and expect value of 10.
An oligotargeter may also be modified by conjugation to a detectable moiety, such as a fluorophore. For example, the 5′ end of a oligotargeter polynucleotide sequence may be conjugated to an FAM dye whose fluorescence can be quenched by gold nanoparticles.
Different oligotargeter sequences and primers (when an amplification step is used) are employed to detect different organisms.
Acinetobacter: Oligotargeters that specifically target genus and species conserved regions in 16s, 23s, ITS, OXA gene, gyrA gene, rpoB gene, aminoglycoside acetyltransferases (AAC), phosphotransferases (APH), and nucleotidyltransferases (ANT) regions will be designed and tested in the AuNPs-based assay. In case of the PCR AuNP based assay, appropriate primers will be designed to amplify each of the above mentioned regions.
Methicillin-resistant staphylococcus aureus (MRSA): Six primers are designed so that the right-junction sequences of SCCmec (types I, II, III, IV and V; one primer for each type) and the conserved sequence orfX gene (highly conserved open reading frame in S. aureus10, 53) are amplified during PCR reaction. The oligotargeter sequences are designed so that they detect conserved regions within the PCR product. In case of direct detection of unamplified MRSA DNA, the assay is performed in two reactions. The first reaction confirms that the causative organism is S. aureus (AuNP-based assay performed using two oligotargeters targeting conserved sequence of orfX gene) and the second determines the presence of MRSA (using two oligotargeters against conserved region within MexA gene).
Hepatitis B virus (HBV): Two primers are designed against regions previously reported to be conserved; namely 1450-1469 in pre-S2 region and 1657-1679 in the S region (numbering takes CTTTTTC of X ORF as start point). The oligotargeters are designed against the conserved region (1571-1592 in S region) within the PCR product. These oligotargeters are used in the AuNP assay for both the direct detection of HBV DNA as well as for the detection of HBV amplicons.
HBV genotyping, 8 pairs of genotype-specific oligotargeters are designed targeting HBV polymerase gene. For each sample, 8 reactions (using unmodified AuNPs) are performed; each reaction utilizing 1 pair of oligotargeters specific to a certain genotype.
With regards to antiviral resistance testing, 12 oligotargeter pairs are designed to detect mutations in reverse transcriptase enzyme at codons 80, 173, 180, and 204 (lamivudine resistance) and at codons 181 and 236 (adefovir resistance). Twelve reaction tubes (using unmodified AuNPs) will be performed per sample, each using a pair of oligotargeters specific either to a wild type or to a mutant at codons 80, 173, 180, 204, 181 or 236.
Regarding the determination of Core Promoter/Precore Mutations that are linked to antiviral resistance mutations, 6 oligotargeter pairs are designed to detect mutations in basal core promoter nucleotides (1762 and 1764) and precore codon 28. Six reaction tubes are performed per sample, each using a pair of oligotargeters specific either to a wild type or to a mutant at basal core promoter nucleotides (1762 and 1764) or precore codon 28.
Human immunodeficiency virus (HIV): One oligotargeter that specifically targets a conserved region in the HIV-1 polymerase gene is designed and tested in the AuNPs-based assay. In case of the PCR AuNP based assay, appropriate primers are designed to amplify the above mentioned region using RT-PCR approach and two oligotargeters that detect a conserved region within the amplified product will be used.
Influenza A Virus (H5N1): Concerning the direct detection of H5N1 RNA, two reactions will be performed; one using an oligotargeter against the matrix gene to confirm the specimen contains influenza A virus and the other using an oligotargeter against HA gene to detect the H5N1 virus. Concerning the PCR AuNP based detection, primers are designed to perform two RT-PCR reactions for each sample; one for the amplification of the matrix gene and the other to amplify the HA gene. Two oligotargeters that recognize a conserved region within each of the two amplified regions (matrix gene and HA gene) will be used.
Target region describes the portion of the Acinetobacter, mycobacteria, staphylococcus, HBV, HIV, Influenza or West Nile virus nucleic acids to which the oligotargeter binds. This target region may lie in a conserved or unique region of the nucleic acid of the pathogen or on a portion unique to a particular genus, species, strain or subtype. The target region is not limited to protein-encoding sequences, but may encompass other portions of a pathogen's genomic or cellular nucleic acids, including control or regulatory sequences or introns or non-transcribe or non-translated nucleic acids.
Nucleic acid generally refers to DNA or RNA or their chemical derivatives. It encompasses nucleic acids isolated from a pathogen as well as amplified nucleic acids or cDNA. It also encompasses modified or mutated nucleic acids, such as variants of a known nucleic acid sequence containing one or more single nucleotide polymorphisms, or more generally, those having a sequence containing 1, 2, 3, 4, 5 or more insertions, deletions, transpositions, or substitutions to a known sequence.
Reaction buffer is one that permits or facilitates interaction of an oligotargeter, a target nucleic acid sequence, and gold nanoparticles. Exemplary buffers include phosphate buffer saline, and other buffers used in PCR reaction mixtures.
Citrate buffer is one containing citrate that can be used to prepare or suspend the colloidal gold nanoparticles (AuNPs). Alternatively, a buffer containing hydrazine, L-tryptophan, an alcohol, especially a lower C1-C6 alcohol, an ether, or sodium diphenyl aminosulfonate may be used. A preferred salt is trisodium citrate salt at a concentration of 30-50 mM or 1-2 wt % (no specific pH). Suitable buffers and methods for making and using colloidal gold are incorporated by reference to John Turkevich. Colloidal gold. Part I. Gold Bull. 1985; 18(3): 86-92; John Turkevich. Colloidal gold. Part II. Gold Bull. 1985; 18(4):125-131; and Katherine C. Graber, R. Grissith Freeman, Micheal B. Hommer, Micheal J. Natan. Preparation and characterization of gold colloid monolayers. Analytical Chemistry 1995; 67(4): 735-743.
Fluorometric detection refers to a method in which a fluorescent dye, such as a fluorescein derivative like FAM (Fluorescein amidite) dye or other fluorophore, has been conjugated to the 5′end of the oligotargeter sequence as described above and used to develop a nanoparticle surface energy transfer (NSET)-based detection assay. For example, an FAM molecule is quenched in the absence of target nucleic acid by the gold nanoparticles, while in the presence of target nucleic acids, hybridization occurs between the oligotargeter and the target RNA and so, the polynucleotide sequence is detached from the gold nanoparticles and hybridizes to the target complementary sequence. FAM emission becomes detectable and indicates a positive sample.
Kit refers to a composition of matter containing one or more ingredients necessary to practice the method of detecting mycobacteria according to the invention. Preferably, the kit will contain gold nanoparticles and a polynucleotide that binds to target nucleic acids in separate containers. A kit may also contain at least one biological sample preservative or additive for a sample, such as an agent that prevents degradation of DNA or RNA, a DNA or an RNA extractant buffer for extracting, isolating or purifying target nucleic acid from a sample, a reaction buffer in which gold nanoparticles, the polynucleotide binding to target nucleic acids and the biological sample are mixed, a negative control sample, a positive control sample, one or more reaction containers, such as tubes or wells, a colorimetric chart, a packaging material, an instruction for use in detecting Acinetobacteria, mycobacteria or the other pathogens described herein.
A subject includes humans, other primates (e.g., a chimpanzee), mammals, birds, reptiles as well as other and other animals susceptible to microbial infection.
SNPs: Single Nucleotide Polymorphisms.
SPR: Surface Plasmon Resonance.
Designing and Verification of Primers and Oligotargeters
The selection and design of primers and oligotargeters were done according to the following approach.
Sequence Retrieval
The target organisms whole genome sequence of the representative strains were retrieved from the NCBI gene bank database as (genebankfull) file format.
The whole genome files were used for testing the designed primers and targeters in silico.
Target gene sequences representing different strain was retrieved from NCBI database in FASTA format.
Multiple Sequence alignment: AlignX Vector NTI Advance® v. 11.5
Target genes from different strain were aligned using alignX.
Multiple sequence alignment was examined for conserved region, GC % content and Tm
Genomic regions showed high similarity and conservation among strains in addition to suitable GC content and melting temperature were chosen for oligotargeter design.
Primers and Oligotargeter Specificity
After selecting conserved regions and regions of difference, proper primers and oligotargeters were selected.
The specificity of primers and oligotargeters were evaluated by blasting them against Nucleotide collection Database (nr/nt) using NCBI blast.
Interference with Human RNA
For pathogens that to be tested in biological fluids such as blood or serum, interference with Homo sapiens genomic material is expected.
The oligotargeter sequence was also blasted against Human genome and transcript database and reference RNA database to evaluate the interference of the human chromosomal element DNA or RNA with the probe functionality.
In Silico PCR
Primers pairs were finally in silico tested for ability to amplify selected targets using NCBI primer blast and Vector NTI.
Selection of Primer Pairs and Oligotargeters for Detection of Microbial Pathogen after PCR-Based Amplification
Different combinations of primer pairs and oligotargeters were tested experimentally by manipulation of reaction conditions and parameters to identify the sequence combinations that can detect the target microbial nucleic acids with highest sensitivity and specificity.
Combination Sets of Primers and Oligotargeters
Primer sets were selected to cover/flank the target gene while oligotargeters were selected to hybridize to the middle region of the target gene. Primers and oligotargeters were selected to have close GC content and annealing conditions and similar length, but minimal secondary structure and no complementary sequences to attempt to optimize amplification and detection strategies. For example, specific primer sets were selected for use in nested, semi-nested, or multiplex PCR reactions. However, after rigorous and systematic assessment of different combinations of sequences, it was surprising that only some combinations of primers and oligotargeters provided high sensitivity and specificity for detection of the particular target pathogens. While not being limited to any particular explanation of these surprising results, factors that may explain success of certain sets to achieve highest specificity and sensitivity include: length of the primer/oligotargeter, sequence conservation of target regions, presence of stable secondary structure of target sequence, number of mismatches in the primers/oligotargeters, number of copies and concentration of target gene, hybridization format (solid phase vs. liquid), stringency of hybridization conditions, and technical strategies implemented to eliminate/reduce cross-hybridization or background noise signals. The tables below describe combinations of primers and oligotargeters for the target pathogens described herein that provide excellent specificity and sensitivity.
Examples of Combinations of Primers and Oligotargeter Sets Conferring Excellent Sensitivity and/or Specificity
Acinetobacter Primers & Oligotargeters
Mycobacteria Primers & Oligotargeters
Methicillin Resistant Staphylococcus Aureus Primers & Oligotargeters
Hepatitis B Virus Primers & Targeter
Influenza a Virus (H5N1) Primers & Oligotargeter
West Nile Virus Primers & Oligortargeter
The Examples below are provided only for illustrative purposes and do not limit the scope of the present invention. Numerous embodiments within the scope of the claims will be apparent to those of ordinary skill in the art, thus the following non-limiting examples only describe particular embodiments of the invention.
Acinetobacter
Acinetobacter spp. are aerobic Gram-negative bacilli commonly present in soil and water as free-living saprophytes. They are also isolated as commensals from skin, throat and various secretions of healthy people, as well as causing human infections (Bergogne-Berezin 2001).
Epidemiology
Strains of Acinetobacter are widely distributed in nature and are found in virtually all samples of soil and fresh water when appropriate culture techniques are used (Bergogne-Berezin and Towner 1996). A range of species have been identified from soil, sewage, plants and food products, although the species associated with human disease are not normally found in such sites.
Human Carriage
Human carriage of Acinetobacter has been demonstrated in normal individuals: it forms part of the bacterial flora of the skin and has been found in the axillae, groin and toe webs of normal individuals. Acinetobacter colonizes the oral cavity, the respiratory tract and the gastrointestinal tract and is found predominantly in moist skin areas (Bergogne-Berezin and Towner 1996)
Sources and Spread in Nosocomial Infections
Sporadic cases of Acinetobacter infections are seen in many hospitals and in a variety of patient settings. However, outbreaks of infection caused by endemic strains are being increasingly described, particularly in ICUs. The carriage rate of Acinetobacter on the skin of hospitalized patients is significantly higher than the community, and this site has been thought to be an important source (Seifert et al., 1997). This has been postulated to be due to reduced hygiene standards among hospitalized patients and the warm, humid atmosphere of hospital beds, which is supported by the observation that colonization is more frequent in summer months. However, two recent studies using DNA-based identification techniques have demonstrated that much of this colonization is due to species of Acinetobacter not commonly associated with clinical infection (Seifert et al., 1997; Berlau et al., 1999)
Laboratory Diagnosis
Isolation
All the frequently encountered species of Acinetobacter grow readily on common laboratory media. In investigating outbreaks, commonly used selective and/or differential media [e.g., MacConkey agar, cysteine lactose electrolyte-deficient (CLED) agar] have been made more selective for particular strains with specific antibiotic resistance patterns by the addition of antibiotics (Allen and Green 1987). Alternatively, a selective medium such as Leeds Acinetobacter medium may be used for the selective isolation of most Acinetobacter spp. (Jawad, Hawkey et al. 1994). When looking for small numbers in environmental specimens, liquid enrichment in minimal media with vigorous shaking has been useful (Bergogne-Berezin and Towner 1996). Although A. baumannii, genomospecies 3 and 3TU have growth optima of 37 degrees C., a lower temperature such as 30 degrees C. will ensure that all species are isolated.
Identification
The differentiation of the different genomospecies is not possible using phenotypic characteristics, although some level of discrimination is possible. Few clinical laboratories have the facilities for the molecular identification of genomospecies and rely on commercial phenotypic systems (e.g. API2ONE [BioMerieux]).
Several studies have shown a poor correlation with DNA-based methods (Bernards, van der Toorn et al. 1996; Jawad, Snelling et al. 1998) Ribotyping (Gerner-Smidt 1992) and rRNA sequencer fingerprinting (Janssen and Dijkshoorn 1996), AFLP (Ehrenstein, Bernards et al. 1996) and ARDRA should all accurately identify individual genomospecies, particularly differentiating 1, 2, 3 and 3TU.
Therapeutic Options for Treatment of Acinetobacter Infection
The therapeutic options for treating Acinetobacter nosocomial infection are restricted because of the high levels of antimicrobial resistance. Based on the results of animal models of Acinetobacter pneumonia (Joly-Guillou, Wolff et al. 1997), carbapenems or carboxypenicillins have been used, in most cases in combination with an aminoglycoside, as an empirical treatment for nosocomial pneumonia. Other strategies have used β-lactamase inhibitors, and among them, sulbactam has demonstrated therapeutic efficacy largely because of its intrinsic activity as a single drug against Acinetobacter (Wood, Hanes et al. 2002).
Materials and Methods for Example 1:
Acinetobacter Samples
A total of 25 clinical samples were obtained (10 pus, 10 sputum, 2 urine, 2 endotracheal tube swap, 1 wound).
Samples Processing and Isolates Identification
Clinical samples were processed and isolated on MacConkey agar media. Isolated colonies were identified by their colony morphology, microscopical examination including gram's and capsule stain, growth at 44° C., biochemical identification and PCR. Biochemical identification tests were Oxidase test, citrate utilization test and Glucose utilization test
PCR Amplification
Multiplex PCR ofb/aOXA-51-like, blaOXA-23-like and class I integrase of Acinetobacter baumannii:
Oxa-23, Oxa-51 and class I integrase gene were simultaneously amplified in a multiplex PCR reaction for the identification of Acinetobacter baumannii. PCR was carried out in 25 μl reaction volumes with 12.5 pmol of each primer, and 1.5 U of TaqDNA polymerase in 1×PCR buffer containing 1.5 mM MgCl2 and 200 μM of each dNTPs. PCR reaction conditions were, 94° C. for 3 min, and then 35 cycles at (94° C. for 45 s, at 57° C. for 45 s, and at 72° C. for 1 min), followed by a final extension at 72° C. for 5 min. See
Amplification of ITS Regions:
PCR of ITS region was performed on extracted DNA and directly isolated colonies (colony PCR). PCR was performed in a total reaction volume of 50 μl consisting of 75 mM Tris-HCl (pH 8.8), 20 mM ammonium sulfate, 1.5 mM MgCl2, 0.8 mM deoxyribonucleoside triphosphates (0.2 mM each), 1 μM (each) primer, and 1 U of Taq DNA polymerase (Fermentas). The PCR performed in 35 cycles: initial denaturation 94° C. for 3 min at 94° C. for 1 min, annealing 55° C. for 1 min, and extension 72° C. for 1.5 min and a final extension step at 72° C. for 7 min. A negative control was included with each test run by replacing the template DNA with sterilized water in the PCR mixture. See
Nested PCR
Nested PCR was performed on purified ITS amplicons. PCR was performed in a total reaction volume of 50 μl consisting of 75 mMTris-HCl (pH 8.8), 20 mM ammonium sulfate, 1.5 mM MgCl2, 0.8 mM deoxyribonucleoside triphosphates (0.2 mM each), 1 μM (each) primer, and 1 U of Taq DNA polymerase (Fermentas). The PCR performed in 35 cycles: initial denaturation 94° C. for 3 min, 94° C. for 1 min, annealing 48° C. for 1 min, and extension 72° C. for 1.0 min and a final extension step at 72° C. for 7 min. Negative control (No template control) was included with each test run.
Gold Nanoparticles Synthesis
Spherical gold colloid was prepared using citrate reduction method. Gold colloid absorption peak was scanned by spectrophotometer in the range of 400-700 nm with a concentration of 14 nM.
AuNPs Detection Probes and Optimization
Detection of PCR Product
Colony PCR was performed on isolated colonies as previously described followed by detection of the amplicon using specific oligotargeters. Detection of PCR product was done by preparing 10 ul reaction containing 52 mM NaCl, 1 μL 100 mM PB, 1.5 μM oligo-targeters and 2 μL PCR product followed by denaturation at 95° C. for 60 s and annealing at 48.9° C. for 60s. Then 30 μL gold colloid 15 nm±2 was added. See
Results for Example 1:
Size & Distribution of the Prepared AuNPs:
Scanning electron microscope image was analysed and the mean particle diameter was found to be 15 nm, and the particles were monodispersed.
Isolates Identifications are shown by the Table of
The blaOXA-5 land blaOXA-23 like Multiplex PCR identification of Acinetobacter baumannii.
Nested PCR.
Assay Prototype: Detection of Acinetobacter in PCR Amplicons is depicted by
AuNPs assay compared to Biochemical, PCR and Nested PCR tests.
Direct Detection of Mycobacterium tuberculosis Complex Using Gold Nanoparticles Specimens
Sputum samples could be cultured on either liquid (7H9 or 7H10 broth) or solid media (Lowenstein Jensen) and processed for DNA extraction. Other biological specimens such as sputum, bone marrow, whole blood, urine, or infected macrophages are treated first for inactivation in category 3 biosafety level and regarded as potential hazardous then processed for DNA extraction.
Clinical specimens are heated in a sealed tube for 80° C. for 1 hour for killing of bacterial cells. Subsequent steps of cell lysis, protein removal, DNA precipitation and recovery are done in class 2 or 3 laminar flow cabinet. All Mycobacterium tuberculosis molecular detection methods require culturing of samples followed by DNA extraction.
DNA Extraction
Briefly, cells were harvested and re-suspended in Tris-EDTA buffer (TE); pH 8.0 and cells were centrifuged and harvested. All cell pellets were stored at −20° C. for a minimum of 4 h. Cell pellets were thawed and suspended in TE buffer pH 8.0. An equal volume of chloroform/methanol (C/M) 2:1 were added and mixed. The suspension was centrifuged where the mycobacteria formed a firm band at the organic-aqueous interface. Tightly bacterial band was left in the tube and both the organic and aqueous layers were decanted. To remove the remaining of the organic solvent, the uncapped tube containing the depilated cells were put in water bath at 55° C. TE buffer pH 8.0 was added and the cells were suspended by vigorous vortex. Tris-HCl; pH 9.0 was added to increase the pH of the cell suspension. Lysozyme (Promega) was added to a final concentration of 100 μg/mL and incubated at 37° C. To remove cell proteins and contaminants, 10% SDS (Promega) and proteinase K (Promega) was added. The extract was mixed by inverting the tubes up and down several times and incubated at 55° C. for 3 h. Proteins were extracted from the resulting suspension by adding an equal volume of phenol/chloroform/isoamyl (P/C/I) 25:24:1 (Sigma Aldrich) and gentle shaking for 30 min followed by centrifugation. The aqueous layer was gently transferred to a sterile tube. To re-extract the aqueous layer, an equal volume of C/I 24:1 was added for 5 min with gentle rocking and repeat the step of centrifugation. To precipitate TB genomic DNA, 3 M sodium acetate (Sigma), pH 5.2, and isopropanol (Sigma) were added. The solution was centrifuged at room temperature to pellet the DNA. The supernatant was discarded and the pellet washed with cold 70% ethanol. The DNA pellet re-suspended in nuclease-free water. Other DNA extraction methods include: silica nanoparticles modified with TB DNA probes, silicon beads, sonication or chemical treatment using 2% SDS-10% Triton-X, guanidinium thiocyanate, NaOH with heating.
Restriction Digestion of DNA
Genomic DNA was digested with a restriction enzyme Bam HI (Promega) by incubation at 37° C. for 1 hour followed by deactivation at 65° C. for 15 min.
PCR Amplification
PCR primers are TBF: ACATGCAAGTCGAACGGAAAGG (SEQ ID NO: 612) and TBR: CCTCCTGATATCTGCGCATTCCAC (SEQ ID NO: 613).
PCR was done using PCR master mix (Promega) and Bam HI digested DNA. BamHI restriction enzyme was selected based on the absence of a restriction site within the specified region. 5% Dimethyl sulfoxide (DMSO, Promega) was added in the master mix. DMSO was added to facilitate TB DNA strand separation. Unidentified mycobacterium specimens were detected first by semi-nested PCR before AuNPs detection. Semi-nested PCR was done on purified amplicons. Genus and species semi-nested PCR used PCR amplicons as a target DNA and employed genus and species oligo-targeters as reverse and forward primers, respectively. Another aim for semi-nested PCR was to prove the specificity of the selected oligo-targeters. To confirm that genus oligotargeter is specific for mycobacterium genus and species oligotargeter is specific for M. tuberculosis complex.
Gold Nanoparticles Concentration.
Spherical gold colloid was prepared using citrate reduction method. The resulted spherical gold nanoparticles were filtered using a 0.45 μm pore size acetate filter (Sigma), and transferred into a clean storage glass bottle. Gold colloid absorption peak was scanned by spectrophotometer in the range of 400-700 nm with a concentration of 8-20 nM.
Oligo-Targeters Selection
Using Two Oligo-Targeters
Genus and species specific hybridization oligo-targeters were selected from previous literature based on a conserved 16s rDNA gene part and synthesized by BIONEER.
Using Single Oligo-Targeter
As an optimization of the assay, species specific hybridization oligotargeter only was used to increase the assay specificity. Other genomic DNA targets can be used for TB diagnosis such as Internal Transcribed Spacer (ITS) and Insertion sequence IS6110 region.
Assay Hybridization Buffer
Detection of PCR product was done by adding NaCl, Tris-HCl buffer; pH 8.
Two Versions of the TB Nano-Gold Assay
Two versions of the nano-gold assay were developed. The first assay detected mycobacterium genus from other bacteria. The second nano-gold method differentiated M. tuberculosis complex from mycobacterium genus using species specific oligotargeter. Both nano-gold prototypes detected TB DNA as PCR amplicons and genomic DNA directly.
Materials and Methods for Example 2
Mycobacterium Samples: For reference strains, two Mycobacterium reference strains of Mycobacterium H37Ra and Mycobacterium smegmatis were cultivated and refreshed on Lowenstein Jensen solid media. A total of 25 clinical DNA samples were obtained, 11 samples are anonymous and 14 samples are identified as Mycobacterium tuberculosis.
Samples Processing
All Mycobacteria reference strains were processed for DNA extraction according to established methods after enzymatic cell lysis.
PCR Amplification
16S DNA PCR
16S DNA PCR was done using PCR master mix (Promega). 5% Dimethyl sulfoxide (DMSO, Sigma-Aldrich) was added in the master mix. PCR was done in 30 cycles: initial denaturation at 95° C. for 2 min, denaturation at 95° C. for 30 s, annealing temperature at 46° C. for 1 min, extension at 72° C. for 45 s, and final extension at 72° C. for 2 min in a thermal cycler (MyCycler, Bio-Rad, California, USA). Genes, PCR primers sequences, Tm and amplicon length is shown by
Semi-Nested PCR
Semi-nested PCR was done on purified 16S rDNA amplicons.
Genus and species semi-nested PCR were done in two separate reactions for each sample. Semi-nested PCR was done in 25 cycles: initial denaturation at 95° C. for 2 min, denaturation at 95° C. for 30 s, annealing temperature at 50° C. for genus and 52° C. for species for 1 min, extension at 72° C. for 45 s, and final extension at 72° C. for 2 min in a thermal cycler (Bio-Rad).
Gold Nanoparticles Synthesis
Spherical gold colloid was prepared using citrate reduction method. Gold colloid absorption peak was scanned by spectrophotometer in the range of 400-700 nm with a concentration of 14 nM.
TB Nano-Gold Assay
Detection of PCR Products
Detection of PCR product was done by adding 44 mM NaCl, 2.5 μL 1M Tris-HCl, 0.02 μM oligo-targeters and 5 μL PCR product followed by denaturation at 95° C. for 30 s and annealing at 48° C. for 30 s. Then 30 μL gold colloid 15 nm±2 was added. Oligotargeter sequences, Blast hits and Alignment identity is shown by
Detection of Genomic DNA
Detection of genomic DNA of 25 clinical strains was done by adding 2.5 μL TrisHCl, 44 mM NaCl (0.5 M), 1 μL species oligotargeter (1 μM), 4 μL genomic DNA, and 3.54 μL H2O. The mixture was denatured at 95° C. for 3 min and annealed at 46° C. for 1 min, then 30 μL AuNPs was added. Positive and negative samples were scanned with a spectrophotometer in the range of 400-700 nm.
Detection Limit of TB Nano-Gold Assay
For PCR products, a two fold serial dilution was done of Mycobacterium H37Ra for a DNA range from 14.1 ng to 0.44 ng. For genomic DNA, a two fold serial dilution was done for genomic DNA of a clinical strain of Mycobacterium tuberculosis complex (range from 40 ng to 10 ng). Dilutions were tested with nano-gold assay with different DNA concentrations as described above.
Results for Example 2
Size & Distribution of the Prepared AuNPs:
Scanning electron microscope image was analyzed and the mean particle diameter was found to be 15 nm, and the particles were monodispersed as shown in
16S rDNA PCR Amplification
PCR for DNA of clinical strains resulted in 16S rDNA target amplification and clear bands of molecular weight about 700 bp were visualized on 0.7% agarose gel electrophoresis.
Semi-Nested PCR Amplification
Semi-nested PCR was done to identify anonymous mycobacterium specimens before AuNPs detection and to prove the specificity of the selected oligotargeters.
Assay Prototype 1: Detection Of MTBC in PCR Amplicons is shown by
Assay Prototype 2: Detection of MTBC in Genomic DNA is shown by
Assay Detection Limits are shown by
Conclusions for Example 2
Unmodified spherical AuNPs were used for direct and quick detection of Mycobacterium tuberculosis complex DNA after PCR amplification. A second version of the assay detected TB DNA directly after restriction digestion of TB genomic DNA isolated from clinical specimens using a single oligotargeter that recognizes 16s rDNA gene segment. The assay detection limit was 1 ng for PCR product and 40 ng for digested genomic DNA. The assay showed 100% sensitivity and 100% specificity as compared with bacterial culture method (gold standard) and semi-nested PCR. The assay turnaround time is about 1 hour including sample digestion and detection of extracted DNA. The TB nano-gold assay is inexpensive and does not require sophisticated instruments or expensive reagents. It represents a promising front line test for diagnosing Mycobacterium tuberculosis complex. Pending further optimization, it can replace the Ziehl-Neelsen staining detection method in developing countries.
Targeting RNA sequences from mycobacteria presents several challenges due to the harsh assay conditions necessary required to lyse Mycobacterium cells and possible sample contamination with host RNA. Mycobacteria RNA sequences are detected under physical cell disruption condition or single step RNA extraction using guanidium thiocynate. This approach is applied to RNA obtained from isolates(colony) identification and to RNA obtained from clinical samples which contain a sufficient concentration of mycobacterial RNA and a sufficiently low concentration of host RNA. Oligotargeters and assay conditions are selected based on the quantity and quality of the RNA sample.
Standard and Clinical Isolates used:
Standard strains:
Clinical Isolates:
Clinical isolates previously identified as mycobacteria tb were obtained from clinical Laboratory and were used to for testing
Mycobacterium tuberculosis strain D (+ve M.TB)
Strain Cultivation and DNA Extraction:
Strain cultivation and DNA extraction performed according to procedures previously described by Parish, et al. [16]. Purified DNA extract was then used as template for amplification of 16s rRNA gene target region.
PCR Amplification of Target Region
Genomic DNA extract was used to amplify the 16s rRNA gene target region using the primers TBF and TBR previously designed. The PCR product was visualized on agarose gel and analyzed the bands size confirmed by gel documentation using Lab Image software. PCR product was purified using Promega PCR clean up kit. M. Tb PCR Conditions are described by
Nested PCR for detection of M. tb DNA. The specificity of the M. tb oligotargeters was confirmed by performing a second PCR using the first PCR as template (nested PCR) and the PCR product was visualized by gel electrophoresis as shown in
PCR Product Quantitation
PCR quantitation of amplicons was done through measuring spectroscopic absorbance for diluted samples ( 1/10) at 260 and 280 nm
DNA was extracted from four different mycobacterial strains. Oligotargeters were added in hybridization buffer containing NaCl and the mixture was denatured, annealed, and allowed to cool to room temperature. AuNPs were then added and change in color was observed in tested specimens as shown in
N=Negative Samples
S=Smegmatis (Non-Mycobacterium complex strain)
R=H37Ra standard strain (Mycobacterium complex Sample)
B, D=clinical isolates (Mycobacterium complex strain)
Detection Limit
Detection limit was determined by performing the assay on serial dilutions of PCR products. The detection limit was initially between 0.6-0.8 ng=0.0028-0.00373 pMol/ul respectively. Results for Example 2C are shown by
After optimizing the assay on PCR template genomic DNA templates were evaluated using Mycobacteria DNA extract. Detection of the 16s rRNA gene target in genomic extract after novel treatment of the genomic extract was successful. Clear color change was observed between positive and negative samples.
Detection of Mycobacterium Tuberculosis DNA Using Gold Nanoparticles
Designing PCR Primers for the Required 16S rRNA Gene Part Using Vector NTI
Vector NTI v11 software was used to analyze the H37Ra sequence and to design PCR Primer to amplify the 16S rRNA gene target region. These primers were designated TBF (forward primer) and TBR (backward primer).
Confirming Specificity of 16SrRNA Gene Probes of Genus and Species Through Blasting on NCBI
For proof of the inventors' concept, oligonucleotides described by Boddinghaus, B., T. Rogall, et al. (1990), Detection and identification of mycobacteria by amplification of rRNA. J. Clin. Microbiol. 28(8): 1751-1759 were used. These oligos were specific at the group, genus or species level and were used to develop a sensitive taxon-specific detection utilizing PCR. Two oligos were selected. The first one was specific to the mycobacteria genus and binds with all mycobacteria species thus differentiating mycobacteria from other strains. The second oligo was species specific differentiating pathogenic mycobacteria complex from non-mycobacterium complex strains. To confirm the specificity of the probes we performed blast against gene bank. These probes were designated TBG (genus probe) and TBS (species Probe). Both probes showed 100% specificity against their targets as shown by Blast results.
Different primers and oligotargeters specific to different conserved regions in the genome Mycobacteria were designed; two overlapping regions in the 16S region, the ITS region, and the IS6110. The primers were designed using Vector NTI Advance® v. 11.5. The conserved regions were chosen based on the multiple alignment analysis performed against Mycobacterium genomes sequences published in NCBI Gene Bank. The determined conserved regions were used to design different specific primer pairs in addition to different oligotargeters. The alignment of the primers with the Mycobacteria genomes was performed using NCBI Nucleotide Blast, while that of the oligotargeters was performed using AlignX Vector NTI Advance® v. 11.5 and NCBI Nucleotide Blast. The oligotargeter sequences were run through a blast analysis against NCBI database to determine assess their specificity to different mycobacterium strains, and the respective degree of homology was determined.
The alignment of the primers with the Mycobacteria genomes was performed using NCBI Nucleotide Blast, while that of the oligotargeters was performed using AlignX Vector NTI Advance® v. 11.5 and NCBI Nucleotide Blast. The oligotargeter sequences were run through a blast analysis against NCBI database to determine assess their specificity to different mycobacterium strains, and the respective degree of homology was determined. selection criteria included:
Different pairs of primers were designed targeting two conserved regions (region 1 and 2) in the 16S region of Mycobacterium genus using Vector NTI Advance® v. 11.5. The conserved regions were chosen based on the multiple alignment analysis performed against Mycobacterium genomes sequences published in NCBI Gene Bank. The determined conserved 16S regions were used to design the six primer pairs in addition to three different oligotargeters. The oligotargeters are specific for sequences within the amplicons produced by any of the primer pairs. Oligotargeters were found to have high degrees of homology only with pathogenic mycobacterium species.
ITS Conserved Region
Three different pairs of primers were designed targeting the conserved ITS region of Mycobacterium genus, in addition to five different oligotargeters. The oligotargeters are specific for sequences within the amplicons produced by any of the three primer pairs. One of the five oligotargeters (MycCompITS.1) is specific to mycobacterium tuberculosis complex, while the others are specific to the mycobacterium genus.
WNV Oligotargeter Design
The inventors have developed specific oligotargeter sequences for identifying WNV using the gold nanoparticle-based methods described herein. These oligotargeter sequences are shown by SEQ ID NOS: 539 (4603-5131 target), 550 (5′UTR), 551-553 (Mat_Peptide_2), 554-555 (Mat_Peptide_1), 556-557 (Mat_Peptide_3), 558-563 ((Mat_Peptide_5), 564-569 (Mat_Peptide_6), 570-571 (Mat_Peptide_7), 572-573 (Mat_Peptide_8), 574-581 (Mat_Peptide_9), 582-583 (Mat_Peptide_10), 584-587 (Mat_Peptide_12), 588-596 (Mat_Peptide_13) and 597-601 (3′UTR).
These sequences were developed using the multiple sequence alignment too AlignX Vector NTI Advance® v. 11.5. Sample results are shown in
GI:315259427, GI:21929238, GI:21929234, GI:21929240, GI:21929236, GI:21929232, GI:21929232, GI:374670387, GI:374670383, GI:374670379, GI:374670375, GI:374670371, GI:374670367, GI:374670363, GI:374670359, GI:374670355, GI:374670351, GI:374670389, GI:374670381, GI:374670377. URL address: http://_www.ncbi.nlm.nih.gov/nuccore?term=west%20nile%20virus%20complete%20genome.
Blast analysis employed the NCBI blast engine http://_www.ncbi.nlm.nih.gov/Database: Human genome and Transcript and Nucleotide (nr/nt), RNA reference sequence.
The multiple sequence alignment was examined for conserved region, GC % content and Tm. Genomic regions showed high similarity and conservation among strains in addition to suitable GC content and melting temperature were chosen for oligotargeter design. Since detection of West Nile Virus will take place in serum samples interference with Homo sapiens genomic material is expected. Selected conserved regions were then blasted against Nucleotide collection Database (nr/nt) using NCBI blast to evaluate oligotargeter specificity. The oligotargeter sequence was also blasted against human genome and transcript database and reference RNA database to evaluate the interference of the human chromosomal element DNA or RNA with the probe functionality. PCR primers were evaluated by NCBI Primer blast.
An example of how WNV virus oligotargeter sequences were developed is shown in
A colloidal solution of AuNPs with a diameter of 15 nm±2 was prepared by citrate reduction of hydrogen tetracloroaurate (III) (HAuCl4. 3H2O) as described elsewhere [16]. Briefly, the reflux system was cleaned by aqua regia and then rinsed with ultrapure water, and blown out with N2. An aqueous solution of HAuCl4.3H2O (1 mM, 100 mL) was brought to reflux while stirring, then 10 mL of 1% trisodium citrate (38.8 mM) were added quickly. This resulted in consequent change in solution color from yellow to clear to black to purple to deep red. Afterwards, the solution was refluxed for an additional 15 minutes and then allowed to cool to room temperature. The colloidal solution was then filtered through 0.45 μm acetate filter, and transferred into a clean storage glass bottle.
The size and distribution of the prepared AuNPs were characterized using field emission scanning electron microscopy (Model: Leo Supra 55). One drop of the AuNPs solution was added onto a silicon slide that was allowed to air dry before examination. The λmax for AuNPs was measured using UV spectrophotometer (Jenway 6800). The concentration of the prepared AuNPs was calculated as described previously, which is incorporated by reference.
Sample Type & Nucleic Acid Extraction
Acinetobacter
Different types of samples will be collected from infected patients (Acinetobacter identified phenotypically on selective media and/or PCR-positive) and normal controls (non-infected healthy volunteers, Acinetobacter identified phenotypically on selective media and/or PCR negative) including urine, sputum, and whole blood. DNA extraction will be performed using either commercial DNA extraction kit or standard DNA extraction procedures. In both cases, DNA extraction procedures steps will include; cell lysis, protein removal, DNA precipitation and recovery, and finally DNA resuspension.
Mycobacterium tuberculosis
Various kinds of samples may be collected including sputum and other bacterial isolates. DNA extraction will be performed using either commercial DNA extraction kit or standard DNA extraction procedures. In both cases, DNA extraction procedures steps will include; cell lysis, protein removal, DNA precipitation and recovery, and finally DNA resuspension.
Methicillin-Resistant Staphylococcus Aureus (MRSA)
Different types of samples will be collected from infected patients/carriers (MRSA was identified phenotypically on selective media and antibiogram and/or PCR-positive) and normal controls (non-infected healthy volunteers, MRSA identified phenotypically on selective media and antibiogram and/or PCR negative) including swabs from anterior nares, throat, perineum, rectum and wounds and sputum of ventilated patients. Swabs will be taken using sterile cotton or Dacron swabs that will be placed in a liquid buffered medium for transportation to the laboratory. Samples should be refrigerated while transported and analyzed within 5 days. Bacterial cells from swabs will be lysed and genomic DNA extracted using QIAGEN Genomic DNA extraction kit according to manufacturer's instructions38. Overnight enrichment (culture) will be performed in samples prior to lysis if needed.
Hepatitis B Virus (HBV)
Serum samples from HBV PCR positive patients and healthy individuals (HBV PCR negative) will be collected, transported (refrigerated) and stored at −80° C. for a maximum of 5 days prior to DNA extraction. DNA extraction will be performed using QIAamp DNA Blood Mini Kit (Qiagen) according to manufacturer's protocol.
Human Immunodeficiency Virus (HIV)
Serum samples from HIV PCR positive patients and healthy individuals (HIV PCR negative) will be collected, transported (refrigerated) and stored at −80° C. for a maximum of 5 days prior to DNA extraction. Viral RNA will be extracted using Promega SV total RNA isolation system using a modified protocol.
Influenza A (H1N1 & H5N1)
The most appropriate sample for testing for influenza virus is from upper respiratory tract e.g. nasal or nasopharyngeal swabs. Swabs will be collected from infected birds/humans (H5N1, H1N1; PCR-positive) and normal controls (PCR negative). Samples can be stored for up to 72 hours at 4° C., and ideally, they should be tested within 24 hours of collection. For long term storage beyond 72 hours, samples should be stored −70° C.31. Viral RNA will be extracted using Promega SV total RNA isolation system using a modified protocol.
West Nile Virus (WNV)
Serum and cerebrospinal fluid can be used for the detection of WNV.
Microbial nucleic acids were extracted using colloidal silica nanoparticles conjugated to an oligonucleotide specific to the target nucleic acid. First, 200 nm colloidal silica nanoparticles were synthesized with a modified Stober method. Briefly, absolute ethanol, deionized water, concentrated ammonia and tetraethyl ortho-silicate (TEOS) were mixed and stirred at room temperature for about 1 hour. Then, the formed colloidal solution was centrifuged at 4,000 rpm for 10 minutes, and the supernatant was discarded and the pellet was washed with absolute ethanol. This washing step was repeated for about 4 times or until no ammonia odor in the solution. The pellet was then dispersed in absolute ethanol and sonicated for about 5 minutes to remove any aggregates. The produced silica nanoparticles were examined using Scanning Electron Microscope (SEM), to get the morphology and the diameter of the prepared silica nanoparticles.
The prepared silica nanoparticles were functionalized with Amino Propyl Trimethoxy Silane (APMS) to introduce amino groups on the surface of the silica nanoparticles. Briefly, 1 ml of APMS was added to 20 ml of the prepared colloidal silica nanoparticles and stirred at room temperature for at least 2 hours, then the solution was centrifuged at 4,000 rpm for 10 minutes, and the supernatant was discarded and the pellet resuspended in phosphate buffer saline (PBS 1×). The number of silica nanoparticles per ml was calculated. Briefly, one mL of the prepared silica colloidal solution was taken centrifuged, and the supernatant was discarded, then the pellet was dried till complete dryness. The dried pellet was then weighted in milligrams and from the volume taken (1 ml) and the weight obtained, concentration of the colloidal solution has been calculated which is 12 mg/ml. The diameter of the silica nanoparticles was measured to be about 150 nm. The weight of one particle equals volume of the particle*specific gravity (2.3), the volume equals 4/3 π r3, and the particles count equals concentration (12 mg/ml)/weight of one particle equals 2.95125E+12 silica nanoparticles/mL.
Synthesis of Silica Probes
To prepare silica probe, heterobifunctional cross linker (3-maleimidobenzoic acid N-hydroxyl succinimide, MBS) that has NHS ester at one end which reacts with primary amine groups to form stable amide bond, The other end has maleimide group which reacts with sulfhydryl groups, therefore the cross linker bound to the amine functionalized silica nanoparticles through the NHS ester and to thiolated probe through the maleimide group and HCV specific probe conjugated to silica nanoparticles was prepared. The thiol labeled probe was prepared as previously described. 10 mg of MBS dissolved in 1 ml dimethyl formamide plus 2.5 ml PBS and 2.5 ml of amino functionalized silica nanoparticles and mix at room temperature for at least 2 hours, and then purified by centrifugation, the thiol modified probe was added to the MBS conjugated silica nanoparticles and incubate at room temperature for at least 2 hours. The number of probes per one silica nanoparticle was calculated by first multiplying the number of moles of the probe by Avogadro's number, and then dividing the number of probes calculated by the silica nanoparticles count, and it was about 500 probes per one silica nanoparticle.
Extraction of Microbial Nucleic Acids from Clinical Samples Using the Prepared Silica Probes
To 200 μL of clinical specimen, 200 μL of lysis buffer (Promega SV viral RNA) was added. After mixing by inversion, 50 μL Proteinase K (or other reagents according to the type of the clinical specimens) was added and left to incubate for 10 min. The mixture was heated to 95° C. in a heat block for 2 min then 50 μL silica-probes was added and the reaction mixed for 1 hr. The mixture was centrifuged at 3000 RPM for 3 min and the pellet was washed twice with nuclease-free water. The target nucleic acids were then eluted by heating at 95° C. for 5 min. The mixture was centrifuged and the supernatant contained the eluted nucleic acid was separated. The extracted nucleic acid was tested using both Real-time RT-PCR and the developed colorimetric AuNP-based assay.
Listing of Sense, Antisense and Oligotargeter Sequences
Acinetobacter Primers & Oligotargeters
16S Region: Primers & Oligotargeters
Acinetobacter ITS Region: Primers & Oligotargeters
Acinetobacter 23S Region: Primers & Oligotargeters
5S rRNA region with intragenic region between 23S and 5S
Primers & Oligotargeters
Insertion Sequence ISAba1, Complete Sequence; and OXA-23 Carbapenemase (Oxa-23) Gene
Primers & Oligotargeters
Mycobacteria Primer and Oligotargeter Sequences
16s rRNA Region: Primers & Probes
Mycobacteria ITS Region: Primers & Probes
ITS Region: Primers & Probes
Methicillin Resistant Staph Aureus (Mrsa) Primers & Oligotargeters
16S rRNA Region: Primers & Probes
MRSA 23S rRNA Region: Primers & Probes
MRSA ITS rRNA Region: Primers & Probes
MRSA mecAgene: Primers & Probes
MRSA femA gene: Primers & Probes
MRSA gyrA gene: Primers & Probes
MRSA spagene: Primers & Probes
Hepatitis B Virus Primers & Oligotargeters
Primers & Oligotargeters:
Human Immunodeficiency Virus 1: Primers and Oligotargeters
Human Immunodeficiency Virus 2: Primers and Oligotargeters
Influenza A Virus (H5N1) Primers & Oligotargeters
Hemagglutinin Gene: Primers & Oligotargeters
Influenza Neuraminidase Gene: Primers & Oligotargeters
Influenza Matrix protein (M1) gene: Primers & Oligotargeters
West Nile Virus Primers & Oligotargeters
Gold Nanoparticles:
Mycobacterium Tuberculosis (TB):
Acintobacter:
Mecithillin-Resitant Staph Aureus (MRSA):
Hepatitis B Virus (HBV):
Human Immunodeficiceny Virus (HIV):
Avian Infleunza Virus (AIV):
West Nile Virus (WNV)
Each document, patent, patent application or patent publication cited by or referred to in this disclosure is incorporated by reference in its entirety, especially with respect to the specific subject matter surrounding the citation of the reference in the text. However, no admission is made that any such reference constitutes background art and the right to challenge the accuracy and pertinence of the cited documents is reserved.
This application claims priority to U.S. Provisional Application No. 61/473,238, filed Apr. 8, 2011 and to U.S. Provisional Application No. 61/473,242, filed Apr. 8, 2011, which are both incorporated by reference in their entireties.
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| Number | Date | Country | |
|---|---|---|---|
| 20140356859 A1 | Dec 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61473238 | Apr 2011 | US | |
| 61473242 | Apr 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14110639 | US | |
| Child | 14294011 | US |
