Patent Application
20110086338
The invention pertains to combining quantum dots (QDs) with engineered phage for the specific identification of biological targets including bacterial strain(s) or cells from clinical or environmental isolates.
The number and diversity of bacteriophages in the environment provide a promising natural pool of specific detection tools for pathogenic bacteria. Currently there are several methods for detection of pathogenic bacteria that exploit phage (McKinstry, M. & Edgar, R. in Phages: Their Role in Bacterial Pathogenesis and Biotechnology, eds. Matthew, K.-W., Friedman, D.-I., & Adhya, S.-L. (ASM Press, Washington, D.C. 2005), pp. 430-440): a plaque assay for detection of Mycobacterium tuberculosis (McNerney, R. et al. 2004 J Clin Microbiol 42:2115-2120); fluorescence labeled phage and immunomagnetic separation assay for detection of Escherichia coli (E. coli) O157:H7 (Goodridge, L. et al. 1999 Int J Food Microbiol 47:43-50; Goodridge, L. et al. 1999 Appl Environ Microbiol 65:1397-1404); phage-based electrochemical assays (Neufeld, T. et al. 2003 Anal Chem 75:580-585); a luciferase reporter mycobacteriophage and Listeria phage assays (Banaiee, N. et al. 2001 J Clin Microbiol 39:3883-3888; Loessner, M.-J. et al 1996 Appl Environ Microbiol 62:1133-1140); and detection of the phage-mediated bacterial lysis and release of host enzymes (e.g., adenylate kinase) (Blasco, R. et al. 1998 J Appl Microbiol 84:661-666).
Two limiting features when detecting pathogenic bacteria are sensitivity and rapidity. Common fluorophores (e.g., green fluorescence protein and luciferase) used as reporters have two major disadvantages: low signal-to-noise ratio due to auto-fluorescence of clinical samples and of bacterial cells and low photo-stability such as fast photobleaching. To overcome these disadvantages, we employed new fluorescent semiconductor nanocrystals, quantum dots (QDs) (Sukhanova A. et al. 2004 Anal Biochem 324:60-67). QDs are colloidal semiconductor (e.g., CdSe) crystals of a few nanometers in diameter. They exhibit broadband absorption spectra and their emissions are of narrow bandwidth with size-dependent local maxima. The presence of an outer shell of a few atomic layers (e.g., ZnS) increases the quantum yield and further enhances the photostability resulting in photostable fluorescent probes superior to conventional organic dyes. Recently, development in surface chemistry protocols allows conjugation of biomolecules onto these QDs to target specific biological molecules and probe nano-environments (Dubertret, B. et al. 2002 Science 298:1759-1762; Yao, J. et al. 2005 Proc Nati Acad Sci USA 102:14284-14289; Hahn, M.-A. et al. 2005 Anal Chem 77:4861-4869). The power to observe and trace single QDs or a group of bio-conjugated QDs, enabling more precise quantitative biology, has been claimed to be one of the most exciting new capabilities offered to biologists today (Michalet, X. et al. 2005 Science 307:538-544; Tokumasu, F. et al. 2005 J Cell Sci 118:1091-1098).
Typically, the detection of small numbers of bacteria in environmental or clinical samples requires an amplification step involving the growth of bacteria in culture to increase cell number. This procedure considerably prolongs the detection time, especially for slow growing bacteria. Here we report a sensitive, rapid and simple method for detection of bacteria. This method combines in vivo biotinylation of engineered host-specific bacteriophage and conjugation of the phage to streptavidin-coated quantum dots. This phage-based assay reduces the “amplification” to a short time (5 to 20 min from infection to lysis) since each infected bacterium can result in a release of 10-1000 phage that can be readily detected by the use of QDs.
The invention is related to a non-biotinylated bacteriophage that comprises a nucleic acid sequence encoding a biotinylation domain.
The invention is also related to a complex that comprises a biotinylated bacteriophage, and a biotin-specific ligand conjugated bioconjugate.
The invention is further related to a method of detecting a bacterial cell in a sample comprising contacting the sample with a non-biotinylated bacteriophage that comprises a nucleic acid sequence encoding a biotinylation domain, wherein the bacteriophage is specific to the bacterial cell.
With current concerns of antibiotic resistant bacteria and bioterrorism, it has become important to rapidly identify infectious bacteria. Traditional technologies are often time consuming, involving isolation and amplification of the causative agents. Rapid and simple methods that can be employed to detect any desired target bacteria would be of great utility. We report a new, rapid and simple method that combines in vivo biotinylation of engineered host-specific bacteriophage and conjugation of the phage to streptavidin-coated quantum dots. The method provides detection and identification of as few as 10 bacterial cells/ml in experimental samples, including about one hundred fold amplification of the signal in one hour. We believe that the method can be applied to any bacteria susceptible to specific phages and would be particularly useful for detection of bacterial strains that are slow growing, e.g., Mycobacterium, or are highly infectious, e.g., Bacillus anthracis. We also envision simultaneous detection of different bacterial species in the sample as well as for applications in studying phage biology.
The preferred embodiment centers on combining quantum dots (QDs) with engineered phage for the specific identification of biological targets including bacterial strain(s) or cells from clinical or environmental isolates. The novel combination of phage quantitatively labeled with QDs will enable the detection and quantification of low abundance targets present down to the single copy level. This preferred embodiment includes the following two aspects, (1) the method to combine phage-quantum dot complex, and (2) the idea to use this complex to target biological samples including bacteria strain(s).
In a time of bio-terrorism threats it is necessary to have new methods available for specifically detecting biological samples such as bacterial pathogens. Different challenges need to be addressed when trying to identify pathogenic bacteria in the ambient, non-laboratory situation. A detection system needs to be rapid, highly sensitive, and specific. The preferred embodiment centers on combining quantum dots (QDs) with engineered bacteriophage for the specific identification of target biological sample(s) including bacterial strain(s) from clinical or environmental isolates.
The unique optical characteristics of QDs such as photostability, size-dependent spectral properties on the same nanometer scale as its linked bacteriophage makes the combination highly suitable for imaging and discovery of single pathogenic bacteria. The combined system has the potential to overcome current limitations of conventional fluorophore-based methods to detect pathogenic bacteria. Current detections utilize optical and electrochemical measurement of nucleic acid or peptide sequences bound to organic fluorophore dyes. The current organic dyes have significant limitations including (1) photobleaching and (2) narrow excitation and spectral overlap in multiplexed detection. For a recent review see Gao X., Yang L., Petros A. J., Marshall F. F., Simons W. J., and Nie S., 2005, Current Opinion in Biotechnology 16:1-10.
The preferred embodiment combines functionalized (e.g., surface-coated with specific proteins, peptides, etc.) QDs with modified bacteriophage engineered to express surface-coat-molecule(s) capable of specifically binding to the functionalized QD. The modified bacteriophage are also adapted to be highly specific to target biological samples, for instance, strains of bacteria. The novel combination of phage quantitatively labeled with QDs will enable the detection and quantification of low abundance targets present down to the single copy level. Unlike organic fluorescence dyes, QDs are stable, non-diluting, non-bleaching, and they are fluorescence emitters covalently and quantitatively linked to phage.
This preferred embodiment includes the following two aspects, (1) the method to combine phage-quantum dot complex (phage-QD) and (2) the idea to use this complex to target bacteria strain(s). The procedure to practice the preferred embodiment is summarized as follows:
(1) Screen, select, and harvest specific phage targeting a specific target including bacterial strain(s) using standard biomolecular methods.
(2) Engineer the harvested phage to have the major head, capsid protein assembly of the phage express the binding sites of specific molecule(s) such as biotin ligase.
(3) Functionalize QDs with specific moiety such as streptavidin molecules capable of recognizing/binding the molecule(s) expressed on the engineered phage.
(4) The isolated and engineered phage (described in (1) and (2)) binds selectively to a target receptor on the biological target such as bacteria. The functionalized QDs (described in (3)) then covalently and quantitatively bind to the phage.
(5) The highly specific linkage of QD to the engineered phage and to the biological target such as bacteria (QD-phage-bacteria complex) provides a unique fluorescent signal at the single copy sensitivity.
(6) Single-color or multi-color, multiplexed detections of a variety of biological targets including bacterial strains are done using different QD-phage complexes. Such detections include high-throughput screening of low abundance bacterial strains using one or more of the methods involving microscopy, spectroscopy, and fluorescence flow cytometry. The preferred embodiment is amenable to applications using portable hand-held instruments.
Currently most advanced techniques to detect bacteria rely on the labeling of targets with green fluorescence protein (GFP) gene. For instance Oda et al. used GFP genes to express and fluorescently detect E. coli (M. Oda et al. 2004 Applied and Environmental Microbiology 70:527-534). This procedure requires laboratory equipment and expertise in molecular biology techniques. In addition, the difficulty in detecting bacteria with this approach occurs when the expression of GFP is low, requiring high cost and high-sensitivity fluorescence detection techniques such as single molecule imaging. Furthermore, the GFP photobleaches rapidly, allowing fluorescence measurement only a few seconds to a minute at ordinary microscopy conditions. When the minimum number of phage per bacteria to cause infection (multiplicity of infection, (M.O.I.)) is only a few, bacterial detection with GFP expression will be very difficult due to low fluorescence signal and fast photobleaching rate of GFP. However, the new method will provide the following advantages over the competing method relying on GFP-expression.
(1) Stable and economical optical detection of biological targets such as bacteria strains: Not only are QDs resistant to photobleaching, allowing for extended observation periods, but also QDs are up to 20 times brighter than traditional organic fluorophores, a result of high quantum yield and a large molar extinction coefficient. Our preferred embodiment, based on QDs will enable the detection of phage-bacterium interaction at the single copy level without the tedious efforts such as preparation of ultra-clean substrates and extreme rejection of background fluorescence signal. The target detection protocol is so simple that it can be done at a non-laboratory situation at a very low cost.
(2) Identifying multiple biological targets: The narrow emission band (the typical full width half maximum of 20 nm) allows for high spectral resolution. With the wide selection of emission wavelengths and the high spectral resolution, multiplexed experiments with various Phage-QDs are possible. QDs additionally have broad excitation spectra. This allows for the concurrent excitation of various QDs with a single excitation source. Meanwhile, the series of phage species may be selected or designed to target specific biological targets. These specific phage species can be combined with QDs of certain sizes having distinct colors to enable multiplexed detection using binding specificity between the phage and the targets.
Our preferred embodiment proves that this method will be of immediate use for the detection of a variety of biological targets such as bacterial strains, tumor cells, and other biomimetic targets. Such detections include high-throughput screening of low abundance bacterial strains using one or more of the methods involving microscopy, spectroscopy, and fluorescence flow cytometry.
This detection method can also be adapted to on-site detection of biological targets such as deadly pathogenic bacteria such as O157:H7 E. coli which occasionally causes massive meat product recalls, human illness and death. In one recent outbreak, 18 million pounds of meat were recalled because there was no high sensitivity detection method available. We also envision a generic method for quantitative detection of deadly pathogenic bacteria for the potential economic benefit of U.S. dairy and meat industries as well as bio-threat agent detection at a very low cost.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. See, e.g., Singleton P and Sainsbury D., in Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons, Chichester, N.Y., 2001; Madigan M. T. Martinko J. M. and Parker J. in Biology of Microorganisms, 9th ed., Prentice-Hall, Inc., Upper Saddle River, N.J., 2000, and Fields Virology 4th ed., Knipe D. M. and Howley P. M. eds, Lippincott Williams & Wilkins, Philadelphia 2001.
The following compositions and methods offer an important improvement to existing methods for the detection and quantification of bacterial cells, including food pathogens, such as Listeria, E. coli, Salmonella, and Campylobacter, and medical pathogens, such as Bordetella pertussis, Chlamydia pneumoniae, and Mycoplasma pneumoniae.
The methods of the present invention provide high detection sensitivity in a short time without the need for traditional biological enrichment. For example, the present methods can provide for the detection or quantification of less than about 100, less than about 50 or less than about 10 bacterial cells in a sample. The present methods can provide for the detection or quantification of less than about 5, less than about 4, less than about 3, or less than about 2 bacterial cells in a sample. The methods of the present invention can provide for the detection and quantification of a single bacterial cell in a sample.
The methods of the present invention allow for the rapid detection and quantification of bacterial cells. For example, the methods of the present invention can be performed in less than about ten hours to less than about twelve hours, in less than about four hours to less than about three hours, and in about two hours or less.
The methods of the present invention can accommodate a wide range of samples sizes. For example, samples as large as about 25 grams (gm) or about 25 milliliters (ml) may be used. Samples of about 1 gram (gm) or about 1 ml or less may be used. If necessary, prior to an assay, samples may be concentrated to reduce the sample volume.
Any bacterial cell for which a bacteriophage that is specific for the particular bacterial cell has been identified can be detected by the methods of the present invention. Those skilled in the art will appreciate that there is no limit to the application of the present methods other than the availability of the necessary specific phage/target bacteria. Bacterial cells detectable by the present invention include, but are not limited to, bacterial cells that are food pathogens. Bacterial cells detectable by the present invention include, but are not limited to, all species of Salmonella, all species of E. coli, including, but not limited to E. coli O157:H7, all species of Listeria, including, but not limited to L. monocytogenes, and all species of Campylobacter. Bacterial cells detectable by the present invention include, but are not limited to, bacterial cells that are pathogens of medical or veterinary significance. Such pathogens include, but are not limited to, Bacillus spp., Bordetella pertussis, Campylobacter jejuni, Chlamydia pneumoniae, Clostridium perfringens, Enterobacter spp., Klebsiella pneumoniae, Mycoplasma pneumoniae, Salmonella typhi, Staphylococcus aureus, and Streptococcus spp. Cultures of all bacterial cells can be obtained, for example, from American Type Culture Collection (ATCC, P.O. Box 1549, Manassas, Va., USA). Bacterial cells detectable by the present invention also include, but are not limited to, contaminating bacterial cells found in systems of commercial significance, such as those used in commercial fermentation industries, ethanol production, antibiotic production, wine production, etc. Such pathogens include, but are not limited to, Lactobacillus spp. and Acetobacter spp. during ethanol production. Other examples of bacteria include those listed in W. Levinson et al., Medical Microbiology & Immunology, McGraw-Hill Cos., Inc., 6th Ed., pages 414-433 (2000). All bacterial cultures are grown using procedures well known in the art.
The range of bacterial cells to be detected is limited only by host ranges of available bacteriophages. Of particular interest are pathogenic bacteria which are capable of contaminating food and water supplies and are responsible for causing diseases in animals and man. Such pathogenic bacteria will usually be gram-negative, although the detection and identification of gram-positive bacteria is also a part of the present invention. A representative list of bacterial hosts of particular interest (with the diseases caused by such bacterial hosts) includes Actinomyces israelii (infection), Aeromonas hydrophila (gastroenteritis, septicemia), Bacillus anthracis (Anthrax: cutaneous, pulmonary), Bacillus subtilis (not considered pathogenic or toxigenic to humans, animals, or plants), Bacteriodes caccae (anaerobic infection), Bacteriodes distasonis (anaerobic infection), Bacteriodes merdae (anaerobic infection), Bacteriodes ovatus (anaerobic infection), Bacteriodes vulgatus (anaerobic infection), Bacteroides fragilis (anaerobic infection), Bacteroides thetaiotaomicron (anaerobic infection), Bordetella pertussis (Whooping cough), Borrelia burgdorferi (Lyme Disease), Brucella abortus (Brucellosis-cattle), Brucella canis (Brucellosis-dogs), Brucella melitensis (Brucellosis-sheep and goats), Brucella suis (Brucellosis-hogs), Burkholderia pseudomallei (infection: acute pulmonary, disseminated septicemic, nondisseminated septicemic, localized chronic suppurative), Campylobacter coli (diarrhea), Campylobacter fetus (bacteremia), Campylobacter jejuni (fever, abdominal cramps, and diarrhea, Guillain-Barre syndrome), Chlamydia trachomatis (Chlamydia), Clostridium botulinum (botulism), Clostridium butyricum (neonatal necrotizing enterocolitis, NEC), Clostridium difficile (NEC), Clostridium perfringens (myonecrosis-gas gangrene), clostridial cellulites, clostridial myositis, food disease, NEC), Clostridium tetani (tetanus), Corynebacterium diphtheriae (diptheria), Enterococcus durans (infection), Enterococcus faecalis (nosocomial infection), Enterococcus faecium (nosocomial infection), Erysipelothrix rhusiopathiae (erysipelothricosis), Escherichia coli (inflammatory or bloody diarrhea, urinary infection, bacteremia, meningitis), Francisella tularensis (tularemia), the genus Fusobacterium (anaerobic infection), Haemophilus aegyptius (mucopurulent conjunctivitis, bacteremic Brazilian purpuric fever), Haemophilus aphrophilus (bacteremia, endocarditis and brain abscess), Haemophilus ducreyi (chancroid venereal disease), Haemophilus influenzae (bacterial meningitis, bacteremia, septic arthritis, pneumonia, tracheobronchitis, otitis media, conjunctivitis, sinusitis, acute epiglottitis, endocarditis), Heaemophilus parainfluenzae (bacteremia, endocarditis and brain abscess), Helicobacter pylori (gastric and duodenal ulcers, gastric cancers), Klebsiella pneumoniae (respiratory, urinary infection), Legionella pneumonphila (Legionaire's disease), the genus Leptospira (leptospirosis, or infectious spirochetal jaundice), Listeria ivanovii (listeriosis), Listeria monocytogenes (listeriosis), Listeria seeligeri (listeriosis), Morganella morganii (infection), Mycobacterium africanum (tuberculosis), Mycobacterium avium-intracellulare (Lady Windermere syndrome, mycobacterium avium complex, MAC), Mycobacterium bovis (tuberculosis), Mycobacterium chelonei (infection), Mycobacterium fortuitum (infection), Mycobacterium kansasii (infection), Mycobacterium leprae (leprosy), Mycobacterium marinum (infection), Mycobacterium tuberculosis (tuberculosis), Mycobacterium ulcerans (infection), Mycobacterium xenopi (infection), Neisseria gonorrhoeae (gonorrhea), Neisseria meningitidis (meningitis), Nocardia asteroids (nocardiosis), Prevotella melaminogenica (anaerobic infection), Proteus mirabilis (infection), Proteus mysofaciens (infection), Proteus vulgaris (infection), Providencia alcalifaciens (infection), Providencia rettgeri (infection), Providencia stuartii (infection), Pseudomonas acidovorans (nosocomial infection), Pseudomonas aeruginosa (nosocomial infection, i.e. in cystic fibrosis patients, burn victims, patients with permanent catheters), Pseudomonas fluorescens (nosocomial infection), Pseudomonas paucimobilis (nosocomial infection), Psuedomonas putida (nosocomial infection), Rickettsia rickettsii (Rocky Mountain spotted fever), Salmonella anatum (gastroenteritis, septicemia), Salmonella bovismorbficans (gastroenteritis, septicemia), Salmonella choleraesuis (gastroenteritis, septicemia), Salmonella Dublin (gastroenteritis, septicemia), Salmonella enteritidis (gastroenteritis, septicemia, enteric fever, bacteremia), Salmonella hirschfeldii (enteric fever), Salmonella Newington (gastroenteritis, septicemia), Salmonella paratyphi (paratyphoid), Salmonella schottmulleri (gastroenteritis, septicemia), Salmonella shottmuelleri (enteric fever), Salmonella typhi (typhoid fever), Serratia marcescens (wound infections), Shigella boydii (shigellosis), Shigella dysenteriae (shigellosis), Shigella flexneri (shigellosis), Shigella sonnei (shigellosis), Spirillum minus (rat-bite fever), Staphylococcus aureus (infections, food poisoning, toxic shock syndrome, pneumonia, bacteremia, endocarditis osteomyelitis enterocolitis, subcutaneous abscesses, exfoliation, meningitis), Streptobacillus moniliformis (rat-bite fever), Streptococcus agalactiae (neonatal sepsis, postpartum sepsis, endocarditis, and septic arthritis), Streptococcus antinosus (invasive infections), Streptococcus bovis (bacterial endocarditis), Streptococcus constellatus (invasive infections), Streptococcus iniae (cellulitis and invasive infections), Streptococcus intermedius (invasive infections), Streptococcus mitior (bacterial endocarditis), Streptococcus nutans (endocarditis), Streptococcus pneumoniae (pneumonia, acute otitis media, infection of the paranasal sinuses, acute purulent meningitis, bacteremia, pneumococcal endocarditis, pneumococcal arthritis, pneumococcal peritonitis), Streptococcus pyogenes (pharyngitis, tonsillitis, wound and skin infections, septicemia, scarlet fever, pneumonia, rheumatic fever and glomerulonephritis), Streptococcus salivarius (bacterial endocarditis), Streptococcus sanguis (bacterial endocarditis), Treponema palladuni (syphilis), Vibrio alginolyticus (diarrhea, infection), Vibrio cholerae (cholera), Vibrio hollisae (diarrhea, infection), Vibrio mimicus (diarrhea, infection), Vibrio parahaemolyticus (diarrhea, infection), Vibrio vulnificus (diarrhea, infection), and Yersinia pestis (plague). The invention may also be used to detect subspecies of bacteria, for example E. coli O157:H7.
In February 2002, the National Institute of Allergy and Infectious Diseases (NIAID) convened the first Blue Ribbon Panel on Bioterrorism and Its Implications for Biomedical Research. This panel of experts was brought together by NIAID to provide objective expertise on the Institute's future counter-bioterrorism research agenda for anthrax, smallpox, botulism, plague, tularemia, and viral hemorrhagic fevers, the pathogens commonly referred to as CDC Category A agents (Table 1). On Oct. 22 and 23, 2002, the NIAID convened another Blue Ribbon Panel of experts to provide objective expertise on the Institute's future biodefense research agenda, as it relates to the NIAID Category B and C Priority Pathogens (Table 1). As a result of these meetings and deliberations a research agenda was developed and widely distributed to the scientific community. One of the goals in managing pathogenic bacteria is to develop improved diagnostics. The initial clinical signs and symptoms of many agents considered biothreats are nonspecific and resemble those of common infections. Therefore, the ability to rapidly identify the introduction of a bioterrorism bacteria or toxin will require diagnostic tools that are highly sensitive, specific, inexpensive, easy to use, and located in primary care settings. Environmental detection is also an important aspect of disease prevention and control.
Bacillus anthracis (anthrax)
Clostridium botulinum (botulism)
Yersinia pestis (plague)
Francisella tularensis (tularemia)
Burkholderia pseudomallei (melioidosis)
Coxiella burnetii (Q fever)
Brucella species (brucellosis)
Burkholderia mallei (glanders)
Shigella species
Salmonella species
Listeria monocytogenes
Campylobacter jejuni
Yersinia enterocolitica
Bacteriophage, also called phage, are highly selective for their hosts. Bacteriophage typing is useful at the species and strain level for identifying bacteria, for instance, in epidemiological investigation of food-borne illness. The specificity of a phage for its host is determined at two levels. Each phage has a host receptor that for tailed phage typically recognizes elements of the phage baseplate and phage tail fibers. Interaction of these components with complementary elements on the bacterial cell surface determines the ability of the phage to bind to the cell and inject its DNA. Enzymatic activity of baseplate elements is sometimes but not always required. There is substantial evidence that phage breeding, genetic engineering of fiber elements, and hybridization, can alter phage specificity at this level. The second level of control over specificity is the events occurring within the bacterial cell, after injection of the phage DNA. Factors that can impact the phage's effectiveness include the presence of restriction enzyme systems in the host and the presence or absence of corresponding protective modifications of the phage DNA, the presence of immunity repressors, and the ability of phage promoters and accessory proteins to co-opt the host RNA polymerase to make phage proteins. Immunity repressors result from the presence of closely related integrated prophages in the target genome and are typically of narrow specificity. Restriction systems and promoter specificity have similar effects on phage expression and plasmid expression, the latter being fairly well understood.
Besides exhibiting specificity, phages have the ability to produce a substantial amplification in a short time. Under optimum infection and host growth medium conditions, a given phage/bacterium combination gives rise to a consistent number of phage progeny. Generally, the lytic infection cycle produces 100 or more progeny phage particles from a single infected cell in about one hour. However, there are exceptions. For example, phi29 of B. subtilis is a premier phage system for study of morphogenesis because it gives a burst of 1,000 in a 35-minute life cycle. Bacteria can be multiply infected by phages (multiplicity of infection, m.o.i.), and the phage “burst” (progeny produced per cell) depends on the multiplicity. To produce high yields an m.o.i. of 10 is generally used. Within an assay it may be necessary to include control comparison standards, done in the same medium, with known numbers of phages infecting known numbers of substrate-bound target cells.
For the detection of a given bacterial cell, a bacteriophage that is capable of infecting the bacterial cell, replicating within the bacterial cell and lysing the bacterial cell is selected. For any given bacterial cell a wide variety of bacteriophages are available, for example, from ATCC or by isolation from natural sources that harbor the host cells. The bacteriophage should also exhibit specificity for the bacterial cell. A bacteriophage is specific for a bacterial cell when it infects the given bacterial cell and does not infect bacterial cells of other species or strains. For the detection of a particular bacterial cell, one would also preferably select a bacteriophage that gives an optimal or maximal burst size.
The range of bacterial cells that can be detected by the present invention is limited only by the availability of a bacteriophage specific for the bacterial cell and will be realized to be vast by those skilled in the art. For example, a searchable database of bacteriophage types available from ATCC is on the worldwide web at atcc.org. Other such depositories also publish equivalent data in their catalogues, and this may be used to identify possible bacteriophage reagents for the methods of the present invention.
Examples of specific bacteria/bacteriophage pairings include PP01, which is specific for E. coli O157:H7 (see, Oda M. et al. 2004 Appl Envir Microbial 70:527-534); phiA1122, which is specific for Yersinia Pestis (see, Garcia E. et al. 2003 J Bacterial 185:5248-5262); D29, which is specific for Mycobacterium tuberculosis (see, McNerney R. et al. 2004 J Clin Microbial 42:2115-2120); T4, which is specific for E. coli (Molecular Biology of Bacteriophage T4, ed. Karam, J. D. (Am. Soc. Microbiol., Washington, D.C.)); and Listeria monocytogenes phage A511, which is specific for L. monocytogenes (see, Loessner et al. 1996 Appl and Environ Microbial 62:1133-1140). Over fourteen different Campylobacter phages are available from ATCC. A number of these are specific for C. jejuni and C. coli and form the basis for a bacteriophage typing system (Grajewski B A et al. 1985 J Clin Microbial 22:13-18). ATCC lists over twenty-four different phages specific for Salmonella; included is phi29, a well-studied phage for Salmonella typhimurium (Zinder, N. D. and Lederberg, J. 1952 J Bacteriology 64:679-699).
High titer bacteriophage stocks are produced on an appropriate host cell strain by procedures well known in the art. For example, plate or broth lysis methods may be used in the production of high titer stocks of bacteriophage. The culture of many other bacteria/bacteriophage pairings is well known to those of skill in the art. See, for example, U.S. Pat. Nos. 5,679,510; 5,914,240; 5,985,596; 5,958,675; 6,090,541; and 6,355,445. See also, for example, Bacteriophages, Mark Adams, InterSciences Publishers, Inc., New York, (1959).
Samples include, but are not limited to, environmental or food samples and medical or veterinary samples. Samples may be liquid, solid, or semi-solid. Samples may be swabs of solid surfaces. Samples may include environmental materials, such as the water samples, or the filters from air samples or aerosol samples from cyclone collectors. Samples may be of meat, poultry, processed foods, milk, cheese, or other dairy products. Medical or veterinary samples include, but are not limited to, blood, sputum, cerebrospinal fluid, and fecal samples and different types of swabs.
Samples may be used directly in the detection methods of the present invention, without preparation or dilution. For example, liquid samples, including but not limited to, milk and juices, may be assayed directly. Samples may be diluted or suspended in solution, which may include, but is not limited to a buffered solution or a bacterial culture medium. A sample that is a solid or semi-solid may be suspended in a liquid by mincing, mixing or macerating the solid in the liquid. A sample should be maintained within a pH range that promotes bacteriophage attachment to the host bacterial cell. A sample should also contain the appropriate concentrations of divalent and monovalent cations, including but not limited to Na+, Mg++, and K+. Preferably a sample is maintained at a temperature that maintains the viability of any pathogen cells contained within the sample.
Biotin (vitamin H), an essential coenzyme synthesized by plants and most procaryotes, is required by all organisms. In cells, biotin in its physiologically active form is covalently attached at the active site of a class of important metabolic enzymes, the biotin carboxylase and decarboxylases.
Biotin protein ligase (BPL), also known as holocarboxylase synthetase, is the enzyme responsible for the covalent attachment of biotin to the cognate proteins. Biotin is attached post-translationally by BPL via an amide linkage to a specific lysine residue of newly synthesized carboxylases in a two-step reaction (
Although the occurrence of biotin-dependent enzymes is ubiquitous in nature, biotinylation is a relatively rare modification in the cell, with between one and five biotinylated protein species found in different organisms (Cronan J. E. Jr. 1990 J Biol Chem 265:10327-10333). Thus, biotin ligase catalyzes a reaction of stringent specificity. The functional interaction between BPL and its protein substrate shows a very high degree of conservation throughout evolution because biotinylation will occur when the two proteins come from widely divergent biological sources (Cronan J. E. Jr. 1990 J Biol Chem 265:10327-10333; Leon-Del-R10 et al. 1995 Proc Natl Acad Sci U.S.A. 92:4626-4630; MacAllister and Coon 1966 J Biol Chem 241:2855-2861; Tissot et al. 1996 Biochem J 314:391-395). The best characterized BPL is the multifunctional BirA protein from Escherichia coli.
Proteins that are biotinylated contain a biotinylation substrate sequence domain that is biotinylated by the BPL. It was found that fusion of a biotinylation domain from a naturally biotinylated protein with any protein of interest provided a general method of specifically labeling chimeric proteins with biotin at a single site. Screens of four biased peptide libraries identified a “consensus peptide” for biotinylation. The “consensus sequence” does not represent absolute sequence requirements for biotinylation, but merely a consensus of the peptide libraries screened. The identified sequences, when fused to either the N- or C-terminus of a variety of proteins can be biotinylated either in vitro or in vivo.
A summary of amino acid sequences active in biotin holoenzyme synthetase (BHS)-catalyzed biotinylation of peptide substrates is described by the consensus sequence: L-X2—X3—I—X5—X5—X7—X8—K—X10—X11—X12—X13 (SEQ ID NO: 1), where X2 is any amino acid; X3 is any amino acid except L, V, I, W, F, or Y; X5 is F or L; X6 is E or D; X7 is A, G, S, or T; X8 is Q or M; X10 is I, M, or V; X11 is E, L, V, Y, or I; X12 is W, Y, V, F, L or I; and X13 is any amino acid except D or E.
A 23-residue peptide (MAGGLNDIFEAQKIEWHEDTGGS) (SEQ ID NO: 2) was identified, which, when fused to the N-terminus of maltose binding protein, was identical to the natural biotin carboxyl carrier protein (BCCP) substrate in the biotinylation reaction by BirA. On its own, this same peptide was also identical to the natural substrate in BirA-catalyzed modification. Measurements of biotinylation of a series of truncates of the 23-mer allowed identification of a 14-residue minimal substrate (GLNDIFEAQKIEWH) (SEQ ID NO: 3) that is biotinylated at a rate within two-fold of the natural protein substrate. Although this 14-mer works well, a slightly extended 15-mer, termed the AviTag, (GLNDIFEAQKIEWHE) (SEQ ID NO: 4) is consistently biotinylated at a rate slightly higher than that of the natural substrate.
Fusion of biotinylation domains to proteins works well at either the N terminus or the C terminus of a target protein. At the N-terminus, an ATG initiation codon is necessary, which, for expression in E. coli, may be followed by an Ala or Ser codon to confer proteolytic stability if the N-terminal Met is removed by methionine aminopeptidase. For fusion at the C terminus of a target protein, the biotinylation domain may be connected to the protein through a short Gly-Gly linker, although it is not clear that the linker is necessary. When a biotinylation domain such as AviTag is fused to a location on the protein other than the termini, the result will likely be extremely variable, depending on whether the biotinylation domain is folded in such a way that the biotin protein ligase (e.g., BirA) can recognize it as a substrate.
Cloning of Biotinylation Domain into Phage Capsid Proteins
In the methods of the invention, a nucleic acid sequence encoding a biotinylation domain is fused with the open reading frame of a bacteriophage capsid protein. It will be appreciated by those of skill in the art that, for a given bacteriophage, there may be multiple capsid proteins into which the biotinylation domain may be inserted so that when the capsid protein is biotinylated, progeny phage will display the biotin moiety such that it is accessible to a biotin-specific ligand (e.g., streptavidin).
To insert a biotinylation domain into a capsid protein by standard molecular cloning methods, it is helpful to know the nucleotide sequence of the bacteriophage capsid protein of interest. Table 2 lists bacteriophages, Genbank accession numbers for bacteriophage genomic sequences sequenced to date, exemplary capsid proteins, the amino acid lengths of the capsid proteins, and Genbank accession numbers for the capsid proteins.
Acholeplasma phage L2
Acholeplasma phage MV-L1
Actinoplanes phage phiAsp2
Acyrthosiphon pisum bacteriophage APSE-1
Aeromonas phage 31
Alteromonas phage PM2
Bacillus anthracis phage Cherry
Bacillus anthracis phage Gamma
Bacillus anthracis phage W Beta
Bacillus clarkii bacteriophage BCJA1c
Bacillus phage GA-1 virion
Bacillus phage phi29
Bacillus thuringiensis bacteriophage Bam35c
Bacillus thuringiensis phage GIL16c
Vibrio phage Vf33
Bordetella phage BIP-1
Bordetella phage BMP-1
Bordetella phage BPP-1
Burkholderia cenocepacia phage Bcep1
Burkholderia cenocepacia phage BcepB1A
Burkholderia cenocepacia phage BcepMu
Burkholderia cepacia complex phage BcepC6B
Burkholderia cepacia phage Bcep176
Burkholderia cepacia phage Bcep22
Burkholderia cepacia phage Bcep43
Burkholderia cepacia phage Bcep781
Burkholderia cepacia phage BcepNazgul
Burkholderia pseudomallei phage phi52237
Chlamydia phage 2
Chlamydia phage 3
Chlamydia phage 4
Chlamydia phage PhiCPG1
Chlamydia phage phiCPAR39
Chlamydia psittaci bacteriophage chp1
Haemophilus phage HP1
Haemophilus phage HP2
Lactobacillus bacteriophage phi adh
Lactobacillus casei bacteriophage A2
Lactobacillus johnsonii prophage Lj928
Lactobacillus johnsonii prophage Lj965
Lactobacillus plantarum bacteriophage LP65
Lactobacillus plantarum bacteriophage phiJL-1
Lactococcus lactis bacteriophage TP901-1
Lactococcus lactis bacteriophage ul36
Lactococcus phage BK5-T
Lactococcus phage P335
Lactococcus phage c2
Listeria bacteriophage P100
Listeria phage 2389 (Bacteriophage PSA)
Listonella pelagia phage phiHSIC
Methanobacterium phage psiM2
Methanothermobacter wolfeii prophage psiM100
Mycobacterium D29
Mycobacterium phage L5
Mycoplasma arthritidis bacteriophage MAV1
Propionibacterium phage phiB5
Pseudomonas aeruginosa bacteriophage PaP2
Pseudomonas aeruginosa phage F116
Pseudomonas aeruginosa phage PaP3
Pseudomonas bacteriophage phi-13 segment L
Pseudomonas bacteriophage phi-13 segment M
Pseudomonas bacteriophage phi-13 segment S
Pseudomonas phage D3
Pseudomonas phage PP7
Pseudomonas phage Pf1
Pseudomonas phage Pf3
Pseudomonas phage gh-1
Pseudomonas phage phi-6 segment L
Pseudomonas phage phi-6 segment M
Pseudomonas phage phi-6 segment S
Pseudomonas phage phiEL
Pseudomonas phage phiKZ
Ralstonia phage p12J
Salmonella typhimurium bacteriophage ES18
Salmonella typhimurium bacteriophage ST104
Salmonella typhimurium bacteriophage ST64T
Salmonella typhimurium phage ST64B
Shigella flexneri bacteriophage V
Sinorhizobium meliloti phage PBC5
Spiroplasma phage 1-C74
Spiroplasma phage 1-R8A2B
Spiroplasma phage 4
Staphylococcus aureus bacteriophage PVL
Staphylococcus aureus phage phi 11
Staphylococcus aureus phage phi 12
Staphylococcus aureus phage phi 13
Staphylococcus aureus phage phiP68
Staphylococcus aureus prophage phiPV83
Staphylococcus aureus temperate phage phiSLT
Staphylococcus phage 44AHJD
Staphylococcus phage K
Staphylococcus phage Twort
Staphylococcus phage phiN315
Streptococcus mitis phage SM1
Streptococcus phage C1
Streptococcus phage Cp-1
Streptococcus pneumoniae bacteriophage MM1
Streptococcus pyogenes phage 315.1
Streptococcus pyogenes phage 315.2
Streptococcus pyogenes phage 315.3
Streptococcus pyogenes phage 315.4
Streptococcus pyogenes phage 315.5
Streptococcus pyogenes phage 315.6
Streptococcus thermophilus bacteriophage 2972
Streptococcus thermophilus bacteriophage 7201
Streptococcus thermophilus bacteriophage DT1
Streptococcus thermophilus bacteriophage Sfi11
Streptococcus thermophilus bacteriophage Sfi19
Streptococcus thermophilus bacteriophage Sfi21
Streptococcus thermophilus temperate
Vibrio cholerae O139 fs1 phage
Vibrio cholerae filamentous bacteriophage fs-2
Vibrio cholerae phage KSF-1phi
Vibrio cholerae phage VGJphi
Vibrio harveyi bacteriophage VHML
Vibrio phage VSK
Vibrio phage Vf12
Xanthomonas campestris pv. pelargonii phage Xp15
Xanthomonas oryzae bacteriophage Xp10
Xanthomonas oryzae phage OP1
Xanthomonas oryzae phage OP2
Xanthomonas phage Cflc
Yersinia pestis phiA1122
The interaction of egg white avidin and bacterial streptavidin with biotin has evolved into an indispensable tool for general use in the biological sciences and as a model for the study of the interaction of a ligand with a protein. Both avidin and streptavidin bind biotin with an essentially immeasurably high affinity constant. The affinity constant for avidin has been estimated at approximately 1015M−1 and that for streptavidin at 1-2 orders of magnitude lower.
The highly specific interaction of avidin with the small vitamin biotin can be a useful tool in assay systems designed to detect and target biological analytes. The extraordinary affinity of avidin for biotin allows biotin-containing molecules in a complex mixture to be discretely bound with avidin conjugates.
Chickens are known to produce several different proteins which bind biotin in a non-covalent fashion. One of them is avidin, which is expressed by oviduct cells upon progesterone induction and is then transferred to the egg-white where it constitutes a minor fraction of the total protein content of the egg-white. Another biotin-binder, called literally biotin-binding protein (BBP), is presumably induced by estrogen and secreted from the liver into chicken plasma. From plasma, the BBP is thought to be deposited in egg-yolk. Another egg-white BBP, distinct from avidin, has biochemical characteristics that resemble those reported for yolk BBP (Seshagiri, P. B. and Adiga, P. R. 1987 Biochim Biophys Acta 926:321-330).
Avidin is a glycoprotein found in the egg white and tissues of birds, reptiles and amphibians. This protein contains four identical subunits having a combined mass of 67,000-68,000 daltons. Each subunit consists of 128 amino acids and binds one molecule of biotin. Avidin is highly glycosylated: carbohydrate accounts for about 10% of the total mass of avidin. Avidin has a basic isoelectric point (pI) of 10-10.5 and is very soluble in water and aqueous salt solutions. Avidin is stable over a wide range of pH and temperature. Extensive chemical modification has little effect on the activity of avidin, making it useful for detection and protein purification.
Streptavidin is another biotin-binding protein that is isolated from Streptomyces avidinii and has a mass of 60,000 daltons. In contrast to avidin, streptavidin has no carbohydrate and has an acidic isoelectric point (pI=5). Streptavidin is much less soluble in water than avidin and can be crystallized from water or 50% isopropyl alcohol. There are considerable differences in the composition of avidin and streptavidin, but they are remarkably similar in other respects. Streptavidin is also a tetrameric protein, with each subunit binding one molecule of biotin with a similar affinity to that of avidin. Guanidinium chloride will dissociate avidin and streptavidin into subunits, but streptavidin is more resistant to dissociation.
Bioconjugate is a generic term to describe detection reagents coupled to proteins, oligonucleotides, small molecules, etc. that are used to direct binding of the detection reagent to an area of interest. Detection reagents (e.g., stains, enzymes and fluorescent nanocrystals) that may be used include, but are not limited to, the fluorescent probe ALEXA (available from Molecular Probes, Inc., Eugene, Oreg.), Cy3, fluorescein isothiocyanate, tetramethylrhodamine, horseradish peroxidase, alkaline phosphatase, glucose oxidase, fluorescent semiconductor nanocrystals (e.g., quantum dots (QDs)) or any other label known in the art. Some proteins for bioconjugation encompass streptavidin, avidin, or protein A.
QD bioconjugate is a generic term used to describe QD nanocrystals coupled to proteins, oligonucleotides, small molecules, etc. which are used to direct binding of the quantum dots to areas of interest. “Qdot®” is a registered trademark belonging to Invitrogen (Quantum Dot Corporation, Hayward, Calif., U.S.A.). Examples of QD bioconjugates include streptavidin, protein A, and biotin families of QD conjugates. QD bioconjugates are often used as simple replacements for analogous conventional dye conjugates when superior performance is required to achieve lower limits of detection, more quantitative results, more photo-stable samples, higher levels of multiplexability, or any of the other advantages afforded by quantum dot technology.
Standard fluorescence microscopes are a useful tool for the detection of QD bioconjugates. These microscopes are often fitted with bright white light lamps and filter arrangements. QD nanocrystals are efficient at absorbing white light using broad excitation filters. Since QD conjugates are virtually completely photo-stable, time can be taken with the microscope to find regions of interest and to adequately focus on the samples. QD conjugates are useful any time bright photo-stable emission is required and are particularly useful in multicolor applications where only one excitation source/filter is available and minimal crosstalk among the colors is required.
To create protein bioconjugates for various assays, a variety of proteins have been either covalently attached or electrostatically self-assembled onto fluorescent semiconductor nanocrystal surfaces (Behrens, S. et al. 2002 Adv Mater 14:1621-1625; Mao, C. B. et al. 2003 Proc Natl Acad Sci USA 100:6946-6951; Chan, W. C. W. & Nie, S. M. 1998 Science 281:2016-2018; Goldman, E. R. et al. 2002 J Am Chem Soc 124:6378-6382; Goldman, E. R. et al. 2002 Anal Chem 74:841-847; Ishii, D. et al. 2003 Nature 423:628-632; Mattoussi, H. et al. 2000 J Am Chem Soc 122:12142-12150; Akerman, M. E. et al. 2002 Proc Natl Acad Sci USA 99:12617-12621; Kloepfer, J. A. et al. 2003 Appl Environ Microbiol 69:4205-4213; Wang, L. Y. et al. 2002 Analyst 127:1531-1534; Lin, Z. B. et al. 2003 Anal Biochem 319:239-243; and Dahan, M. et al. 2003 Science 302:442-445)
In some embodiments of the invention, bacteriophage are engineered to have the major head, capsid protein assembly of the phage express a first attachment site. Additionally a bioconjugate is functionalized with a second attachment site capable of recognizing/binding the first attachment site expressed on the engineered phage. The first attachment site may be a protein, a polypeptide, a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethysulfonylfluoride), or a combination thereof, or a chemically reactive group thereof. The second attachment site of the bioconjugate or reagent that is to be linked to the bacteriophage may be a protein, a polypeptide, a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethysulfonylfluoride), or a combination thereof, or a chemically reactive group thereof.
First and second attachment sites include, but are not limited to, binding pairs such as biotin/streptavidin, antigen/antibody, receptor/ligand partners, protein A/antibody Fc domain, and leucine zipper domains (e.g., JUN-FOS leucine zipper domain).
In one embodiment, a first step is to add non-biotinylated bacteriophage to the test sample. The target bacterial cells are infected when they come into contact with the phage. Infected bacterial cells are incubated under conditions to form biotinylated bacteriophage. Biotinylated bacteriophage may be detected by a variety of means. For example, biotinylated bacteriophage may be detected by contacting the solution with a biotin-specific ligand conjugated bioconjugate. Biotinylated bacteriophage may be concentrated prior to contacting with a biotin-specific ligand conjugated bioconjugate. The presence of biotinylated bacteriophage in the sample indicates the presence of target bacterial cells in the sample and the absence of biotinylated bacteriophage indicates the absence of target bacterial cells in the sample.
In another embodiment, a first step is to add a complex to the test sample in which the complex combines a biotinylated bacteriophage and conjugation of the phage to a biotin-specific ligand conjugated bioconjugate. The target bacterial cells are bound when they come into contact with the complex. The complex may be detected by a variety of means. The presence of complex-bound bacteria in the sample indicates the presence of target bacterial cells in the sample and the absence of complex-bound bacteria indicates the absence of target bacterial cells in the sample.
Preferably throughout detection assays, the sample is maintained at a temperature that maintains the viability of any pathogen cell present in the sample. During steps in which bacteriophage are attaching to bacterial cells, it is preferable to maintain the sample at a temperature that facilitates bacteriophage attachment. During steps in which bacteriophage are replicating within an infected bacterial cell or lysing such an infected cell, it is preferable to maintain the sample at a temperature that promotes bacteriophage replication and lysis of the host. Such temperatures are at least about 25° C., more preferably no greater than about 45° C., most preferably about 37° C. It is also preferred that the samples be subjected to gentle mixing or shaking during bacteriophage attachment, replication and release.
Assays may include various appropriate control samples. For example, control samples containing no bacteriophage or control samples containing bacteriophage without bacteria may be assayed as controls for background levels.
Our strategy of the detection method is shown in
As a model system, we engineered the coliphage T7 to express the major capsid protein, gp10A, fused to the 15-amino acid biotinylation peptide (T7-bio): GLNDIFEAQKIEWHE (SEQ ID NO: 4) (Cull, M.-G. & Schatz, P.-J. 2000 Methods Enzyniol 326:430-440). This cloning strategy (See Example 1 for details) results in the display of the given peptide on all 415 monomers of the major capsid protein. To differentiate the “reagent” phage from phage released from infected target cells, it is crucial that the reagent input phage is not biotinylated. We accomplished this in one of the two ways: (i) by packaging the engineered T7 phage DNA in vitro using wild-type virion proteins or (ii) by propagating our reporter phage on a biotin auxotroph. In the first method we used a commercially available packaging kit composed of wild type phage proteins (T7Select, Novagen). This system when used with the engineered phage T7 DNA gave rise to 103-106 plaque forming units (PFU)/1 μg DNA. In the second method, we prepared phage lysates by two rounds of growth on an E. coli biotin auxotroph that was starved for biotin as was evident by an inhibition of bacterial growth that addition of biotin could relieve. The absence of biotinylated phage resulting from either method of production was confirmed by western blot analysis (using streptavidin-HRP) and fluorescence microscope using streptavidin-QDs (Table 3, last row).
E. coli cells
0
0
0
0
0
0
0
(*1)Based on burst size of 100 phage/cell.
(*2)Only E. coli normalized to 100.
(*3)No phage added.
(*4)Non-biotinylated phage added at the detection step.
The resulting nonbiotinylated “reagent” phage, when making progeny particles following infection of wild type E. coli bacterial cells, gets biotinylated at the displayed peptide by the host BLP resulting in biotinylated phage referred to as T7-bio. As a negative control, we engineered T7 phage to express the major capsid protein fused to the 10-amino acid myc peptide (T7-myc), EQKLISEEDL (SEQ ID NO: 5), which results in a phage that displays the myc peptide but is not recognized by the host BLP.
We tested the ability of the engineered phage to infect E. coli and become biotinylated, by mixing the phage with a bacterial culture until cell lysis was visible. The presence of the biotin molecules on the phage's displayed peptide was initially detected by western blot analysis of the virion proteins from purified phage samples using streptavidin-HRP. The western blot confirmed the in vivo biotinylation of the tagged capsid protein assembled on the phage head (
Biotinylated phage bound to QDs were initially detected and quantitatively analyzed by flow cytometry. Flow cytometry allow scanning of a large number of particles; single cells flow in a fast stream through a focal volume of an excitation laser beam and light intensities due to side scattering (SSC, measured at 90 degrees relative to the direction of the focused laser light), forward scattering (FSC, at 180 degrees), and fluorescence light (FL, at 90 degrees) are monitored. Since phage-QD complexes are too small to be detected by the SSC, we used carrier-cells to which the phage T7 can bind. In
As a second method to detect QD labeled phage, we used fluorescence microscopy, which permits quantitative measurements with the sensitivity to detect a single QD conjugated to a single phage. As with the flow cytometry, we used carrier bacterial cells to allow removal of free QDs by washing.
Detection of E. Coli in a Mix with Other Bacteria and in Environmental Samples
To detect a small number of a given type of bacteria among several different bacterial cells, we used a culture with mixed bacterial strains on which phage T7 cannot propagate: Pseudomonas aeruginosa, Vibrio cholera, Salmonella, Yersinia pseudotuberculosis, and Bacillus subtilis. We analyzed mixtures of 2×106 cells of each of the above strains mixed with different numbers of cells of Escherichia coli (from 10 to 107 cells/ml). We followed the method as illustrated in
Finally, we tested water samples from the Potomac River. We determined, in about one hour, that there are at least 20 E. coli cells in 1 ml of the sample. In comparison, using Coliscan MF kit (Micrology Laboratories, LLC, approved by the US Environmental Protection Agency), it took 24 hours to detect and identify 200 general coliforms in 1 ml of the same samples. The lower number of E. coli cells detected by our method is due to higher specificity of the phage to detect a particular coliform. These results demonstrate the rapid and specific nature of our assay.
The primary significance of the current work is the development of a simple and highly sensitive procedure for phage-based bacterial detection that achieves (i) enhanced detection limit; (ii) rapidity; and (iii) broad applicability. Sensitivity is shown by our method's ability to detect and quantify low abundance targets of at least as few as 10 cells/ml. Our method takes about an hour to get results. The procedure uses biotinylation, a highly conserved pathway in nature, which can be applied to target a variety of bacteria in biological samples. Although we used a single phage-host system, the method may be expanded for the detection of multiple bacterial strains by their specific phages, each conjugated to QDs of different emission colors, in the same sample. Furthermore, higher specificity can be achieved by using multiple phages for one host, in the same sample, conjugated to QDs of different emission colors. The tools for detection can include microscopy, spectroscopy, or flow cytometry. It should be possible to utilize QD phage-based bacterial detection with hand-held instruments. Additionally, since phage could not be seen by light microscopy previously, and QD-labeled phage are infective, we believe that our method opens up new avenues to address phage biology-related questions on topics such as initial binding, phage localization, distribution and more.
We used the T7Select System (Novagen) for engineering and packaging of DNA into T7 phage particles. For the T7-bio we used two phosphorylated primers: 3′L6bio and 5′L6bio, containing overhang sequences for ligation with HindIII and EcoRI digested phage arms DNA, respectively (upper case), a 6 amino acid linker coding sequence (underlined), followed by the biotinylation peptide coding DNA (lower case) and a stop codon (bold):
The primers were annealed to each other by heating at 95° C. for 5 min in ligation buffer and cooling at room temperature, ligation to T7 arms was done as recommended by the manufacturer. For the engineering of the T7-myc phage we used the primers MYC1: 5′-AATTCtggtggcagcggatctgagcagaagctgatcagcgaggaagatcttaattaaA-3′ (SEQ ID NO: 8) and MYC2: 5′-AGCTttaattaagatcttcctcgctgatcagatctgctcagatccgctaccaccaG-3′ (SEQ ID NO: 9) containing overhang sequences for ligation with EcoRI and HindIII digested phage arms DNA (upper case), a 5 amino acid linker coding sequence (underlined), followed by the myc domain (lower case) and a stop codon (bold).
Staining of phage was done as described elsewhere (Palmer, E.-L., & Martin, M.-L. 1988 CRC Press, Inc. Boca Raton, Fla. 154). Briefly, phage were incubated with E. coli for 4 min at 37° C. in PBS buffer. A streptavidin coated QD (QD 605) suspension, 1 μM, (Quantum Dot Corporation, Hayward, Calif., U.S.A.) was diluted 100 fold in PBS. 1 μl of the diluted solution was added and incubation continued for 5 min at room temperature. After centrifugation at 1500 rpm for 5 min, 1 μl of the sample was placed onto a carbon-coated Formvar-filmed copper grid (Tousimis Research Corp. Rockville, Md.) and allowed to attach. The sample was negatively stained with 1%, pH 7.0 phosphotungstic acid solution (Fisher Scientific Co. Fair Lawn, N.J.). The grid was examined by an electron microscope operated at 75 kV (Hitachi 117000, Tokyo, Japan). Digital images were taken by a CCD camera (Gatan Inc. Pleasanton, Calif.).
Phage were incubated with E. coli cells for 4 min at 37° C. 1 μl streptavidin coated Quantum dots (1 μM) were added and incubation continued for 5 min at room temperature. After centrifugation at 1500 rpm for 5 min, 1 μl of the sample was resuspended to 6×104 cells/ml. Samples were analyzed by flow cytometry using BD FACS DNA LSR II (Becton Dickinson) monitoring the ratio of 407/600 nm excitation/emission fluorescence from phage-QDs bound cells. Events shown in histograms were gated on fluorescence. All were detected in log scale, and events were triggered on SSC. A total of 30,000 events were collected for each analysis.
Samples were prepared as described for flow cytometry, except that an additional centrifugation was performed and 2 μl of the sample were placed on a microscope slide, covered with a cover slip and visualized on an Olympus Vanox-T microscope using an Oriel 500 W Hg arc lamp running at 200 W, a fluorescence filter set (a bandpass exciter (447±15 nm), a dichroic mirror (505 nm cutoff), and a longpass emission filter (560 nm cutoff)), and a 1.25 numerical aperture oil immersion objective (DPlan 100×, Olympus). Images were captured by an intensified cooled CCD camera (I-PentaMAX, Roper Scientific, Inc.).
Detection of E. coli in a Mix of Bacteria
2×106 cells of each of the following strains Pseudomonas aeruginosa, Vibrio cholera, Salmonella enterica serovar Typhimurium, Yersinia pseudotuberculosis, Bacillus subtilis were mixed with different numbers of Escherichia coli BL-21 cells, 10-107, as estimated by OD 600 and confirmed by viable count. After about 10-15 min at 37° C., lysates were cleaned by centrifugation and assayed using the fluorescence microscope.
The birA gene was engineered into phage along with a biotinylation domain to allow more of the displayed peptides to be biotinylated, thereby increasing the detection sensitivity. The BPL of E. coli, birA gene, was clone as a transcriptional fusion with the phage Capsid-bio under the phage promoter.
This engineered phage (capsid-bio-birA) had about a 100 fold higher level of biotinylation than the Capsid-bio engineered phage as judged by western blot analysis with streptavidin-HRP. This new engineered phage overcomes potential limitation of the endogenous BirA protein such that most of the displayed biotinylation domain becomes biotinylated.
A fluorescence image of phage-QD complexes' spread on a glass coverslip is shown in
Time-averaged bright field and fluorescence imaging of bacteria cells was done after an attempt to bind maximum number of phage-QD complexes by adding excess number of phage-QD complexes (
Detection of Yersinia pestis with phiA1122
Recombinant, non-biotinylated phiA1122-bio and phiA1122-myc phages are engineered using standard molecular biology protocols. PhiA1122 grows on almost all isolates of Yersinia pestis. Phage are incubated with Y. pestis cells for 4 min at 37° C. Streptavidin coated Quantum dots (1 μM) are added and incubation continued for 5 min at room temperature. After centrifugation at 1500 rpm for 5 min, 1 μl of the sample is resuspended to 6×104 cells/ml. Samples are analyzed by flow cytometry using BD FACS DIVA LSR II (Becton Dickinson) monitoring the ratio of 407/600 nm excitation/emission fluorescence from phage-QDs bound cells. Events shown in histograms are gated on fluorescence. All are detected in log scale, and events are triggered on SSC. A total of 30,000 events are collected for each analysis.
Samples may alternatively be detected by fluorescence microscopy as described in Example 1 and Yersinia pestis is detected in a mix of bacteria as described in Example 1.
Detection of Escherichia coli (O157:H7) with PP01 Bacteriophage
Recombinant, non-biotinylated PP01-bio and PP01-myc phages are engineered using standard molecular biology protocols. The virulent phage PP01 infects E. coli O157:H7 strains with high specificity (Morita M. et al. 2002 FEMS Microbiol Lett 216:243-248). Phage are incubated with E. coli cells for 4 min at 37° C. Streptavidin coated Quantum dots (1 μM) are added and incubation continued for 5 min at room temperature. After centrifugation at 1500 rpm for 5 min, 1 μl of the sample is resuspended to 6×104 cells/ml. Samples are analyzed by flow cytometry using BD FACS DIVA LSR II (Becton Dickinson) monitoring the ratio of 407/600 nm excitation/emission fluorescence from phage-QDs bound cells. Events shown in histograms are gated on fluorescence. All are detected in log scale, and events are triggered on SSC. A total of 30,000 events are collected for each analysis.
Samples may alternatively be detected by fluorescence microscopy as described in Example 1 and E. coli is detected in a mix of bacteria as described in Example 1.
Detection of Mycobacterium tuberculosis by D29
Recombinant, non-biotinylated D29-bio and D29-myc phages are engineered using standard molecular biology protocols. D29 is a lytic, double-stranded DNA phage with a wide mycobacterial host range. Phage are incubated with E. coli cells for 4 min at 37° C. Streptavidin coated Quantum dots (1 μM) are added and incubation continued for 5 min at room temperature. After centrifugation at 1500 rpm for 5 min, 1 μl of the sample is resuspended to 6×104 cells/ml. Samples are analyzed by flow cytometry using BD FACS DIVA LSR II (Becton Dickinson) monitoring the ratio of 407/600 nm excitation/emission fluorescence from phage-QDs bound cells. Events shown in histograms are gated on fluorescence. All are detected in log scale, and events are triggered on SSC. A total of 30,000 events are collected for each analysis.
Samples may alternatively be detected by fluorescence microscopy as described in Example 1 and M. tuberculosis is detected in a mix of bacteria as described in Example 1.
While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, and appendices, as well as patents, applications, and publications, referred to above, are hereby incorporated by reference.
This application claims the benefit of U.S. Provisional Application No. 60/654,784 filed Feb. 18, 2005, which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US06/05537 | 2/16/2006 | WO | 00 | 12/15/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60654784 | Feb 2005 | US |
