Patent Application
20060003352
Throughout this application, various publications are referenced. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Establishing a causal relationship between infection with a virus and a specific disease may be complex. In most acute viral diseases, the responsible agent is readily implicated because it replicates at high levels in the affected tissue at the time the disease is manifest, morphological changes consistent with infection are evident, and the agent is readily cultured with standard microbiological techniques. In contrast, implication of viruses in chronic diseases may be confounded because persistence requires restricted gene expression, classical hallmarks of infection are absent, and/or Methods for cloning nucleic acids of microbial pathogens directly from clinical specimens offer new opportunities to investigate microbial associations in chronic diseases. The power of these methods is that they can succeed where methods for pathogen identification through serology or cultivation may fail due to absence of specific reagents or fastidious requirements for agent replication. Over the past decade, the application of molecular pathogen discovery methods resulted in identification of novel agents associated with both acute and chronic diseases, including Borna disease virus, Hepatitis C virus, Sin Nombre virus, HHV-6, HHV-8, Bartonella henselae, and Tropherema whippeli.
Various methods are employed or proposed for cultivation-independent characterization of infectious agents. These can be broadly segregated into methods based on direct analysis of microbial nucleic acid sequences. (e.g., cDNA microarrays, consensus PCR, representational difference analysis, differential display), direct analysis of microbial protein sequences (e.g., mass spectrometry), immunological systems for microbe detection (e.g., expression libraries, phage display) and host response profiling. A comprehensive program in pathogen discovery would need to exploit most, if not all, of these technologies.
The decision to employ a specific method is guided by the clinical features, epidemiology, and spectrum of potential pathogens to be implicated. Expression libraries, comprised of cDNAs or synthetic peptides, may be useful tools in the event that large quantities of acute and convalescent sera or cerebrospinal fluid are available for screening purposes; however, the approach is cumbersome, labor-intensive, and success is dependent on the presence of a specific, high affinity humoral immune response. The utility of host response mRNA profile analysis has been demonstrated in several in vitro paradigms and some inbred animal models; nonetheless, it is important to formally consider the possibility that a variety of organisms may activate similar cascades of chemokines, cytokines, and other soluble factors that influence host gene expression to produce what are likely to be convergent gene expression profiles. Thus, at least in virology, it is prudent to explore complementary methods for pathogen identification based on agent-encoded nucleic acid motifs. Given the potential for high density printing of microarrays, it is feasible to design slides or chips decorated with both host and pathogen targets. This would provide an unprecedented opportunity to simultaneously survey host response mRNA profiles and viral flora, providing insights into microbial pathogenesis not apparent with either method of analysis alone.
Representational difference analysis (RDA) is an important tool for pathogen identification and discovery. However, RDA is a subtractive cloning method for binary comparisons of nucleic acid populations. Thus, although ideal for analysis of cloned cells or tissue samples that differ only in a single variable of interest, RDA is less well suited to investigation of syndromes wherein infection with any of several different pathogens results in similar clinical manifestations, or infection is not invariably associated with disease. An additional caveat is that because the method is dependent upon the presence of a limited number of restriction sites, RDA is most likely to succeed for agents with large genomes. Indeed, in this context, it is noteworthy that the two viruses detected by RDA in the listing above were herpesviruses.
Consensus PCR (cPCR) has been a remarkably productive tool for biology. In addition to identifying pathogens, particularly genomes of prokaryotic pathogens, this method has facilitated identification of a wide variety of host molecules, including cytokines, ion channels, and receptors. Nonetheless, until recently, a difficulty in applying cPCR to pathogen discovery in virology has been that it is difficult to identify conserved viral sequences of sufficient length to allow cross-hybridization, amplification, and discrimination using traditional cPCR format. While this may not be problematic when one is targeting only a single virus family, the number of assays required becomes infeasible when preliminary data are insufficient to allow a directed, limited analysis.
Real-time PCR methods have significantly changed diagnostic molecular microbiology by providing rapid, sensitive, specific tools for detecting and quantitating genetic targets. Because closed systems are employed, real-time PCR is less likely than nested PCR to be confounded by assay contamination due to inadvertent aerosol introduction of amplicon/positive control/cDNA templates that can accumulate in diagnostic laboratories. The specificity of real time PCR is both a strength and a limitation. Although the potential for false positive signal is low so is the utility of the method for screening to detect related but not identical genetic targets. Specificity in real-time PCR is provided by two primers (each approximately 20 matching nucleotides (nt) in length) combined with a specific reporter probe of about 27 nt. The constraints of achieving hybridization at all three sites may confound detection of diverse, rapidly evolving microbial genomes such as those of single-stranded RNA viruses. These constraints can be compensated in part by increasing numbers of primer sets accommodating various templates. However, because real-time PCR relies on fluorescent reporter dyes, the capacity for multiplexing is limited to the number of emission peaks that can be unequivocally separated. At present up to four dyes can be identified simultaneously. Although the repertoire may increase, it will not likely change dramatically.
This invention provides a method for simultaneously detecting in a sample the presence of one or more of a plurality of different target nucleic acids comprising the steps of:
This invention further provides the instant method, wherein the method detects the presence in the sample of 10 or more, 50 or more, 100 or more, or 200 or more different target nucleic acids. This invention further provides the instant method, wherein the sample is contacted with 4 or more, or 10 or more, or 50 or more, or 100 or more, or 200 or more different primers.
This invention further provides the instant method, wherein one or more primers comprises the sequence set forth in one of SEQ ID NOs:1-96, and 98-101. This invention further provides the instant method, wherein at least two different primers are specific for the same target nucleic acid. This invention further provides the instant method, wherein a first primer is a forward primer for the target nucleic acid and a second primer is a reverse primer for the same target nucleic acid.
This invention further provides the instant method, wherein the mass tags bound to the first and second primers are of the same size. This invention further provides the instant method, wherein the mass tags bound to the first and second primers are of a different size.
This invention further provides the instant method, wherein at least one target nucleic acid is from a pathogen.
This invention further provides the instant method, wherein the presence and size of any cleaved mass tag is determined by mass spectrometry. This invention further provides the instant method, wherein the mass spectrometry is selected from the group consisting of atmospheric pressure chemical ionization mass spectrometry, electrospray ionization mass spectrometry, and matrix assisted laser desorption ionization mass spectrometry.
FIGS. 21A-21B: (A) This figure shows serial dilutions of plasmid standards (5×105, 5×104, 5×103, 5×102, 5×101, and 5×100) for RSV group A, RSV group B, Influenza A, HCoV-SARS, HCoV-229E, HCoV-OC43, and M. pneumoniae were each analyzed by mass tag PCR in a multiplex format. (B) This figure shows simultaneous detection of multiple targets in multiplex format using mixtures of two templates per assay (5×104 copies each): HCoV-SARS and M. pneumoniae, HCoV-229E and M. pneumoniae, HCoV-OC43 and M. pneumoniae, and HCoV-229E and HCoV-OC43.
Terms
As used herein, and unless stated otherwise, each of the following terms shall have the definition set forth below.
“Mass tag” shall mean any chemical moiety (i) having a fixed mass, (ii) affixable to a nucleic acid, and (iii) whose mass is determinable using mass spectrometry. Mass tags include, for example, chemical moieties such as small organic molecules, and have masses which range, for example, from 100 Da to 2500 Da.
“Nucleic acid” shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids thereof. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J., USA).
“Pathogen” shall mean an organic entity including, without limitation, viruses and bacteria, known or suspected to be involved in the pathogenesis of a disease state in an organism such as an animal or human.
“Sample” shall include, without limitation, a biological sample derived from an animal or a human, such as cerebro-spinal fluid, lymph, blood, blood derivatives (e.g. sera), liquidized tissue, urine and fecal material.
“Simultaneously detecting”, with respect to the presence of target nucleic acids in a sample, means determining, in the same reaction vessels(s), whether none, some or all target nucleic acids are present in the sample. For example, in the instant method of simultaneously detecting in a sample the presence of one or more of 50 target nucleic acids, the presence of each of the 50 target nucleic acids will be determined simultaneously, so that results of such detection could be, for example, (i) none of the target nucleic acids are present, (ii) five of the target nucleic acids are present, or (iii) all 50 of the target nucleic acids are present.
“Specific”, when used to describe a primer in relation to a target nucleic acid, shall mean that, under primer extension-permitting conditions, the primer specifically binds to a portion of the target nucleic acid and is extended.
“Target nucleic acid” shall mean a nucleic acid whose presence in a sample is to be detected by any of the instant methods.
“5-UTR” shall mean the 5′-end untranslated region of a nucleic that encodes a protein.
The following abbreviations shall have the meanings set forth below: “A” shall mean Adenine; “bp” shall mean base pairs; “C” shall mean Cytosine; “DNA” shall mean deoxyribonucleic acid; “G” shall mean Guanine; “mRNA” shall mean messenger ribonucleic acid; “RNA” shall mean ribonucleic acid; “PCR” shall mean polymerase chain reaction; “T” shall mean Thymine; “U” shall mean Uracil; “Da” shall mean dalton.
Finally, with regard to the embodiments of this invention, where a numerical range is stated, the range is understood to encompass the embodiments of each and every integer between the lower and upper numerical limits. For example, the numerical range from 1 to 5 is understood to include 1, 2, 3, 4, and 5.
To address the need for enhanced multiplex capacity in diagnostic molecular microbiology we have established a PCR platform based on mass tag reporters that are easily distinguished in Mass Spectrometry (MS) as discrete signal peaks. Major advantages of the PCR/MS system include: (1) hybridization to only two sites is required (forward and reverse primer binding sites) vs real time PCR where an intermediate third oligonucleotide is used (probe binding site); this enhances flexibility in primer design; (2) tried and proven consensus PCR primers can be adapted to PCR/MS; this reduces the time and resources that must be invested to create new reagents and assay controls; (3) the large repertoire of tags allows highly multiplexed assays; additional tags can be easily synthesized to allow further complexity; and (4) sensitivity of real time PCR is maintained. We view PCR/MS as a tool with which to rapidly screen clinical materials for the presence of candidate pathogens. Thereafter, targeted secondary tests, including real time PCR, can be used to quantitate microbe burden and pursue epidemiologic studies.
Specifically, this invention provides a method for simultaneously detecting in a sample the presence of one or more of a plurality of different target nucleic acids comprising the steps of:
In one embodiment of the instant method, the method detects the presence in the sample of 10 or more different target nucleic acids. In another embodiment, the method detects the presence in the sample of 50 or more different target nucleic acids. In a further embodiment, the method detects the presence in the sample of 100 or more different target nucleic acids. In a further embodiment, the method detects the presence in the sample of 200 or more different target nucleic acids.
In one embodiment of the instant method, the sample is contacted with 4 or more different primers. In another embodiment, the sample is contacted with 10 or more different primers. In a further embodiment, the sample is contacted with 50 or more different primers. In a further embodiment, the sample is contacted with 100 or more different primers. In yet a further embodiment, the sample is contacted with 200 or more different primers.
In one embodiment of the instant method, one or more primers comprises the sequence set forth in one of SEQ ID NOs:1-96, and 98-101.
In another embodiment of the instant method, at least two different primers are specific for the same target nucleic acid. For example, in one embodiment a first primer is a forward primer for the target nucleic acid and a second primer is a reverse primer for the same target nucleic acid. In this example, the mass tags bound to the first and second primers can be of the same size or of different sizes. In another embodiment, a first primer is directed to a 5′-UTR of the target nucleic acid and a second primer is directed to a 3D polymerase region of the target nucleic acid.
In one embodiment of the instant method, wherein each primer is from 15 to 30 nucleotides in length. In another embodiment, each mass tag has a molecular weight of from 100 Da to 2,500 Da. In a further embodiment, the labile bond is a photolabile bond, such as a photolabile bond cleavable by ultraviolet light.
In another embodiment of the instant method, at least one target nucleic acid is from a pathogen. Pathogens include, without limitation, B. anthracis, a Dengue virus, a West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, Yellow Fever virus, La Crosse virus, California encephalitis virus, Rift Valley Fever virus, CCHF virus, VEE virus, EEE virus, WEE virus, Ebola virus, Marburg virus, LCMV, Junin virus, Machupo virus, Variola virus, SARS corona virus, an enterovirus, an influenza virus, a parainfluenza virus, a respiratory syncytial virus, a bunyavirus, a flavivirus, and an alphavirus.
In another embodiment, the pathogen is a respiratory pathogen. Respiratory pathogens include, for example, respiratory syncytial virus A, respiratory syncytial virus B, Influenza A (N1), Influenza A (N2), Influenza A (M), Influenza A (H1), Influenza A (H2), Influenza A (H3), Influenza A (H5), Influenza B, SARS coronavirus, 229E coronavirus, OC43 coronavirus, Metapneumovirus European, Metapneumovirus Canadian, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4A, Parainfluenza 4B, Cytomegalovirus, Measles virus, Adenovirus, Enterovirus, M. pneumoniae, L. pneumophilae, and C. pneumoniae.
In a further embodiment, the pathogen is an encephalitis-inducing pathogen. Encephalitis-inducing pathogens include, for example, West Nile virus, St. Louis encephalitis virus, Herpes Simplex virus, HIV 1, HIV 2, N. meningitides, S. pneumoniae, H. influenzae, Influenza B, SARS coronavirus, 229E-CoV, OC43-CoV, Cytomegalovirus, and a Varicella Zoster virus. In a further embodiment, the pathogen is a hemorrhagic fever-inducing pathogen. In a further embodiment, the sample is a forensic sample, a food sample, blood, or a derivative of blood, a biological warfare agent or a suspected biological warfare agent.
In one embodiment of the instant method, the mass tag is selected from the group consisting of structures V1 to V4 of
In another embodiment of the instant method, the presence and size of any cleaved mass tag is determined by mass spectrometry. Mass spectrometry includes, for example, atmospheric pressure chemical ionization mass spectrometry, electrospray ionization mass spectrometry, and matrix assisted laser desorption ionization mass spectrometry.
In one embodiment of the instant method, the target nucleic acid is a ribonucleic acid. In another embodiment, the target nucleic acid is a deoxyribonucleic acid. In a further embodiment, the target nucleic acid is from a viral source.
This invention provides a kit for simultaneously detecting in a sample the presence of one or more of a plurality of different target nucleic acids comprising a plurality of nucleic acid primers wherein (i) for each target nucleic acid at least one predetermined primer is used which is specific for that target nucleic acid, (ii) each primer has a mass tag of predetermined size bound thereto via a labile bond, and (iii) the mass tag bound to any primer specific for one target nucleic acid has a different mass than the mass tag bound to any primer specific for any other target nucleic acid.
This invention also provides a kit for simultaneously detecting in a sample the presence of one or more of a plurality of different target nucleic acids comprising (a) a plurality of nucleic acid primers wherein (i) for each target nucleic acid at least one predetermined primer is used which is specific for that target nucleic acid, (ii) each primer has a mass tag of predetermined size bound thereto via a labile bond, and (iii) the mass tag bound to any primer specific for one target nucleic acid has a different mass than the mass tag bound to any primer specific for any other target nucleic acid; and (b) a mass spectrometer.
This invention further provides a kit for simultaneously detecting in a sample the presence of one or more of a plurality of different target nucleic acids comprising (a) a plurality of nucleic acid primers wherein (i) for each target nucleic acid at least one predetermined primer is used which is specific for that target nucleic acid, (ii) each primer has a mass tag of predetermined size bound thereto via a labile bond, and (iii) the mass tag bound to any primer specific for one target nucleic acid has a different mass than the mass tag bound to any primer specific for any other target nucleic acid, and (b) instructions for use.
Finally, this invention provides a kit for simultaneously detecting in a sample the presence of one or more of a plurality of different target nucleic acids comprising (a) a plurality of nucleic acid primers wherein (i) for each target nucleic acid at least one predetermined primer is used which is specific for that target nucleic acid, (ii) each primer has a mass tag of predetermined size bound thereto via a labile bond, and (iii) the mass tag bound to any primer specific for one target nucleic acid has a different mass than the mass tag bound to any primer specific for any other target nucleic acid; (b) a mass spectrometer; and (c) instructions for simultaneously detecting in a sample the presence of one or more of a plurality of different target nucleic acids using the primers and the mass spectrometer.
This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.
Abbreviations: 5′-UTR, 5′-untranslated region; ALS, Amyotrophic Lateral Sclerosis; APCI, atmospheric pressure chemical ionization; ESI, electrospray ionization; PCR, polymerase chain reaction; MALDI-TOF, matrix assisted laser desorption ionization time of flight; MS, mass spectrometry
Background
Establishing a causal relationship between infection with a virus and a specific disease may be complex. In most acute viral diseases, the responsible agent is readily implicated because it replicates at high levels in the affected tissue at the time the disease is manifest, morphological changes consistent with infection are evident, and the agent is readily cultured with standard microbiological techniques. In contrast, implication of viruses in chronic diseases may be confounded because persistence requires restricted gene expression, classical hallmarks of infection are absent, and/or mechanisms of pathogenesis are indirect or subtle. Methods for cloning nucleic acids of microbial pathogens directly from clinical specimens offer new opportunities to investigate microbial associations in chronic diseases (21). The power of these methods is that they can succeed where methods for pathogen identification through serology or cultivation may fail due to absence of specific reagents or fastidious requirements for agent replication. Over the past decade, the application of molecular pathogen discovery methods resulted in identification of novel agents associated with both acute and chronic diseases, including Borna disease virus, Hepatitis C virus, Sin Nombre virus, HHV-6, HHV-8, Bartonella henselae, and Tropherema whippeli (5-7, 17, 19, 22, 23, 27).
Various methods are employed or proposed for cultivation-independent characterization of infectious agents. These can be broadly segregated into methods based on direct analysis of microbial nucleic acid sequences (e.g., cDNA microarrays, consensus PCR, representational difference analysis, differential display), direct analysis of microbial protein sequences (e.g., mass spectrometry), immunological systems for microbe detection (e.g., expression libraries, phage display) and host response profiling. A comprehensive program in pathogen discovery will need to exploit most, if not all, of these technologies.
The decision to employ a specific method is guided by the clinical features, epidemiology, and spectrum of potential pathogens to be implicated. Expression libraries, comprised of cDNAs or synthetic peptides, may be useful tools in the event that large quantities of acute and convalescent sera or cerebrospinal fluid are available for screening purposes; however, the approach is cumbersome, labor-intensive, and success is dependent on the presence of a specific, high affinity humoral immune response. The utility of host response mRNA profile analysis has been demonstrated in several in vitro paradigms and some inbred animal models (8, 26, 30); nonetheless, it is important to formally consider the possibility that a variety of organisms may activate similar cascades of chemokines, cytokines, and other soluble factors that influence host gene expression to produce what are likely to be convergent gene expression profiles. Thus, at least in virology, it is prudent to explore complementary methods for pathogen identification based on agent-encoded nucleic acid motifs. Given the potential for high density printing of microarrays, it is feasible to design slides or chips decorated with both host and pathogen targets. This would provide an unprecedented opportunity to simultaneously survey host response mRNA profiles and viral flora, providing insights into microbial pathogenesis not apparent with either method of analysis alone. Representational difference analysis (RDA) is an important tool for pathogen identification and discovery. However, RDA is a subtractive cloning method for binary comparisons of nucleic acid populations (12, 18). Thus, although ideal for analysis of cloned cells or tissue samples that differ only in a single variable of interest, RDA is less well suited to investigation of syndromes wherein infection with any of several different pathogens results in similar clinical manifestations, or infection is not invariably associated with disease. An additional caveat is that because the method is dependent upon the presence of a limited number of restriction sites, RDA is most likely to succeed for agents with large genomes. Indeed, in this context, it is noteworthy that the two viruses detected by RDA in the listing above (see first paragraph) were herpesviruses (5, 6). Consensus PCR (cPCR) has been a remarkably productive tool for biology. In addition to identifying pathogens, particularly genomes of prokaryotic pathogens, this method has facilitated identification of a wide variety of host molecules, including cytokines, ion channels, and receptors. Nonetheless, until recently, a difficulty in applying cPCR to pathogen discovery in virology has been that it is difficult to identify conserved viral sequences of sufficient length to allow cross-hybridization, amplification, and discrimination using traditional cPCR format. While this may not be problematic when one is targeting only a single virus family, the number of assays required becomes infeasible when preliminary data are insufficient to allow a directed, limited analysis. To address this issue, we adapted cPCR to Differential Display, a PCR-based method for simultaneously displaying the genetic composition of multiple sample populations in an acrylamide gel format (16). This hybrid method, domain-specific differential display (DSDD), employs short, degenerate primer sets designed to hybridize to viral genes representing larger taxonomic categories than can be resolved in cPCR. The major advantages to this approach are: (i) reduction in numbers of reactions required to identify genomes of known viruses, and (ii) potential to detect viruses less closely related to known viruses than those found through cPCR. The differential display format also permits identification of syndrome-specific patterns of gene expression (host and pathogen) that need not be present in all clinical samples. Additionally, because multiple samples can be analyzed in side-by-side comparisons, DSDD allows examination of the timecourse of gene expression patterns. Lastly, recent experience with isolation of the West Nile virus responsible for the outbreak of encephalitis in New York in the summer of 1999 indicates that DSDD may be advantageous in instances where template is suboptimal due to degradation (e.g., postmortem field specimens).
The development and application of sensitive high throughput methods for detecting a wide range of viruses is anticipated to provide new insights into the pathogenesis of chronic diseases. We are funded through AI51292 to support these objectives by establishing DNA microarray, multiplexed bead-based flow cytometric (MB-BFC) and domain specific differential display (DSDD) assay platforms for viral surveillance and discovery in chronic diseases. Each of these methods has its strengths; however, none is ideal. Microarrays provide a platform wherein one can simultaneously query thousands of microbial and host gene targets but lack sensitivity and are difficult to modify as new targets are identified. Bead-based arrays are flexible but similar in sensitivity to microarrays.
Domain specific differential display is sensitive and flexible but labor intensive. Real time PCR (not a component of our original application but useful to note for purposes of method comparisons), is rapid and sensitive, but cannot be used for broad range detection of viral sequences, because of stringent sequence constraints for the three oligonucleotides comprising the system (two primers, one probe).
Mass-Tag PCR would integrate PCR and mass spectrometry (MS) into a stable and sensitive digital assay platform. It is similar in sensitivity and efficiency to real time PCR but provides the advantages of simultaneous detection and discrimination of multiple targets, due to less stringent constraints on primer selection. Additionally, whereas multiplexing is limited in real time PCR by overlapping fluorescence emission spectra, Mass-Tag PCR allows discrimination of a large repertoire of mass tags with molecular weights between 150 and 2500 daltons.
In Mass-Tag PCR, virus identity is be defined by the presence of label of a specific molecular weight associated with an amplification product. Primers are be designed such that the tag can be cleaved by irradiation with UV light. Following PCR, the amplification product can be immobilized on a solid support and excess soluble primer removed. After cleavage by UV irradiation (˜350 nm), the released tag will be analyzed by mass spectrometry. Detection is sensitive, fast, independent of DNA fragment length, and ideally suited to the multiplex format required to survey clinical materials for infection with a wide range of infectious agents.
Results
Mass spectrometry (MS) is a rapid, sensitive method for detection of small molecules. With the development of new ionization techniques such as matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI), mass spectrometry has become an indispensable tool in many areas of biomedical research. Although these ionization methods are suitable for the analysis of bioorganic molecules, such as peptides and proteins, improvements in both detection and sample preparation will be required before mass spectrometry can be used to directly detect long DNA fragments. A major confound in exploiting MS for genetic investigation has been that long DNA molecules are fragmented during the analytic process. The mass tag approach overcomes this limitation by detecting small stable mass tags that serve as signatures for specific DNA sequences rather than the DNA sequences themselves.
Atmospheric pressure chemical ionization (APCI) has advantages over ESI and MALDI for some applications. Because buffer and inorganic salts impact ionization efficiency, performance in ESI is critically dependent upon sample preparation conditions. In MALDI, matrix must be added prior to sample introduction into the mass spectrometer; speed is often limited by the need to search for an ideal irradiation spot to obtain interpretable mass spectra. APCI requires neither desalting nor mixing with matrix to prepare crystals on a target plate. Therefore in APCI, mass tag solutions can be injected directly. Because mass tags are volatile and have small mass values, they are easily detected by APCI ionization with high sensitivity. The APCI mass tag system is easily scaled up for high throughput operation.
We have established methods for synthesis and APCI analysis of mass tags coupled to DNA fragments. Precursors of four mass tags [(a) acetophenone; (b) 3-fluoroacetophenone; (c) 3,4-difluoroacetophenone; and (d) 3,4-dimethoxyacetophenone] are shown in
DNA Sequencing with Biotinylated Dideoxynucleotides on a Mass Spectrometer
PCR amplification can be nonspecific; thus, products are commonly sequenced to verify their identity as bona fide targets. Here we apply the rapidity and sensitivity of mass tag analyses to direct MS-sequencing of PCR amplified transcripts.
MALDI-TOF MS has recently been explored widely for DNA sequencing. The Sanger dideoxy procedure (25) is used to generate the DNA sequencing fragments. The mass resolution in theory can be as good as one dalton; however, in order to obtain accurate measurement of the mass of the sequencing DNA fragments, the samples must be free from alkaline and alkaline earth salts and falsely stopped DNA fragments (fragments terminated at dNTPs instead of ddNTPs). Our method for preparing DNA sequencing fragments using biotinylated dideoxynucleotides and a streptavidin-coated solid phase is shown in
Sequencing experiments for a 55 bp synthetic template using MALDI-TOF mass spectrometry were recently performed (9). Four commercially available biotinylated dideoxynucleotides ddATP-11-biotin, ddGTP-11-biotin, ddCTP-11-biotin and ddTTP-11-biotin (NEN, Boston) were used to produce the sequencing ladder in a single tube by cycle sequencing. Clean sequence peaks were obtained on the mass spectra, with the first peak being primer extended by one biotinylated dideoxynucleotide. Although the identity of A and G residues were determined unambiguously, C and T could not be differentiated because the one dalton mass difference between the ddCTP-11-biotin and ddTTP-11-biotin cannot be consistently resolved by using the current mass detector for DNA fragments. Nonetheless, these results confirmed that clean sequencing ladders can be obtained by capture/release of DNA sequencing fragments with biotin located on the 3′ dideoxy terminators. The procedure has been improved by using biotinylated ddTTPs that have large mass differences in comparison to ddCTP-11-biotin. Pairing ddTTP-16-biotin (Enzo, Boston), which has a large mass difference in comparison to ddCTP-11-biotin, with ddATP-11-biotin, ddCTP-11-biotin, and ddGTP-11-biotin, allowed unambiguous sequence determination in the mass spectra (
Cloning Viral Targets as Controls for Mass-Tag PCR
Multiple sequence alignment algorithms have been used by our bioinformatics core to extract the most conserved genomic regions amongst the GenBank published enteroviral sequences. Regions wherein sequence conservation meets or exceeds 80% for an enteroviral serogroup or genetically related subgroup have been identified in the 5′-untranslated region (UTR) and the polymerase gene (3D) of the enterovirus genus. A representative collection of virus isolates has been obtained to generate calibrated standards for Mass-Tag PCR (Table 1). The current panel includes 22 isolates representing all characterized serogroups of pathogenic relevance (A, B, C, and D; covering about 90% of all US enterovirus isolates in the past 10 years; the remaining 10% include non-typed isolates). Twelve isolates have been grown and the relevant regions cloned for spotting onto DNA microarrays and use as transcript controls for DSDD, multiplex bead based, and real time PCR assays. Viruses can be propagated in the appropriate cell lines to generate working and library stocks (Rd, Vero, HeLa, Fibroblast, or WI-38 cells). Library stocks can be frozen and maintained in curated collections at −70° C. Viral RNA can be extracted from working stocks using Tri-Reagent (Molecular Research Center, Inc.). Purified RNA can be reverse transcribed into cDNA using random hexamer priming [to avoid 3′ bias] (Superscript II, Invitrogen/Life Technologies).
Target regions of 100-200 bp representing the identified core sequences will be amplified by PCR from cDNA template using virus-specific primers. Products are cloned (via a single deoxyadenosine residue added in template-independent fashion by common Taq-polymerases to 3′-ends of amplification products) into the transcription vector pGEM T-Easy (Promega Corp.). After transformation and amplification in Escherichia coli, plasmids are analyzed by restriction mapping and automated dideoxy sequencing (Columbia Genome Center) to determine insert orientation and fidelity of PCR. Plasmid libraries will be maintained as both cDNAs and glycerol stocks.
Multiple sequence alignment algorithms can be used to identify highly conserved (>95%) sequence stretches of 20-30 bp length within the identified core sequences to serve as targets for primer design.
Synthesis of Primers for Use in Mass-Tag PCR
Highly conserved target regions within the core sequences suitable for primer design are identified by using multiple sequence alignment algorithms adjusted for the appropriate window size (20-30 bp) and conservation threshold (>95%). Final alignments are color-coded to facilitate manual inspection. Parameters implicated in primer performance including melting temperature, 3′-terminal stability, internal stability, and propensity of potential primers to form stem loops or primer-dimers can be assessed using standard primer selection software programs OLIGO (Molecular Biology Insights), Primer Express (PE Applied Biosystems), and Primer Premiere (Premiere Biosoft International). Primers can be synthesized with a primary amine-group at the 5′-end for subsequent coupling to NHS esters of the mass tags (
Sensitivity and Specificity of Mass-Tag PCR for Detection of Enteroviral Transcripts
Although the method disclosed here is useful for detecting viral RNA, plasmid DNA is an inexpensive, easily quantitated sequence target; thus, primer sets can be initially validated by using dilutions of linearized plasmid DNA. Plasmids are selected to carry the viral insert in mRNA sense orientation with respect to the T7 promoter sequence. Plasmids will be linearized by restriction digestion using an appropriate enzyme that cleaves in the polylinker region downstream of the insert. Where the cloned target sequence is predicted to contain the available restriction sites, a suitable unique restriction site is introduced via the PCR primer used during cloning of the respective target. Purified linearized plasmid DNA is serially diluted in background DNA (human placenta DNA, Sigma) to result in 5×105, 5×104, 5×103, 5×102, 5×101, and 5×100 copies per assay.
Once optimal primer sets for detection of all relevant enteroviruses are identified, the sensitivity of the entire procedure including RNA extraction and reverse transcription is assessed. Synthetic RNA transcripts of each target sequence are generated from the linearized plasmid DNA using T7 RNA polymerase. Transcripts are serially diluted in background RNA relevant to the primary hypothesis (e.g., ALS, normal spinal cord RNA). Individual dilutions representing 5×105, 5×104, 5×103, 5×102, 5×101, and 5×100 copies per assay in a background of 25 ng/ul total RNA are extracted with Tri-Reagent, reverse transcribed, and then subjected to Mass-Tag PCR.
Specificity of the identified primer sets relevant to multiplexing can be assessed by using one desired primer set in conjunction with its respective target sequence at 5 times threshold concentration in the presence of all other, potentially cross-reacting, target sequences at a 102-, 104- and 106-fold excess.
PCR amplification is performed using photocleavable mass tagged primers in the presence of a biotinylated nucleotide (e.g. Biotin-16-dUTP, Roche) to allow removal of excess primer after PCR. Amplification products will be purified from excess primer by binding to a streptavidin-coated solid phase such as streptavidin-Sepharose (Pharmacia) or streptavidin coated magnetic beads (Dynal) via biotin-streptavidin interaction.
Molecular mass tags can be made cleavable by irradiation with near UV light (˜350 nm), and the released tags introduced by either chromatography or flow injection into a pneumatic nebulizer for detection in an atmospheric pressure chemical ionization mass spectrometer. Alternatively, to increase the specificity of detection by analyzing only PCR products of the expected size range, the mass tagged amplicons, can be size-selected (without the requirement for biotinylated nucleotides) using HPLC.
Multiplex Detection and Identification of Enteroviral Transcripts
A method that allows simultaneous detection of a broad range of enteroviruses with similar sensitivity was developed. A series of 4 primer sets were identified in the 5′-UTR predicted to detect all enteroviruses. These can be combined into two or perhaps even one mixed set for multiplex PCR. Two different genomic regions, 5′-UTR and polymerase, are targeted with independent primer panels, in order to confirm presence of enterovirus infection.
Once the presence of enteroviral sequences are confirmed using broad range primer sets, a different primer set is used to discriminate amongst the various enteroviral species. Whereas broad range primers are be selected from the highly conserved 5′-UTR and polymerase 3D gene regions, the primer sets used to identify the enterovirus species target the most divergent genomic regions in VP3 and VP1.
Limitations must be considered in that although cerebral spinal fluid is unlikely to contain more than a single enterovirus (the virus responsible for clinical disease in an individual patient), individual stool samples may contain several enteroviruses. It is important, therefore, that assays not favor amplification or detection of one viral species over another. Second, multiplexing can result in loss of sensitivity. Thus, panels should be assessed for sensitivity (and specificity) with addition of new primer sets.
Direct MS-Sequencing of PCR Amplified Enteroviral Transcripts for Virus Species Identification
MALDI MS has been explored widely for DNA sequencing; however, this approach requires that the DNA sequencing fragments be free from alkaline and alkaline earth salts, as well as other contaminants, to ensure accurate measurements of the masses of the DNA fragments. We explored a novel MS DNA sequencing method that generates Sanger-sequencing fragments using biotinylated dideoxynucleotides labeled with mass tags.
The ability to distinguish various nucleotide bases in DNA using mass spectrometry is dependent on the mass differences of the DNA ladders in the mass spectra. Smith et al. have shown that using dye labeled ddNTP paired with a regular dNTP to space out the mass difference can increase the detection resolution in a single nucleotide extension assay (10). Preliminary studies using biotin-11-dd(A, C, G)TPs and biotin-16-ddTTP, indicated that the smallest mass difference between any two nucleotides is 16 daltons. To enhance the ability to distinguish peaks in the sequencing spectra, the mass separation of the individual ddNTPs can be increased by systematically modifying the biotinylated dideoxynucleotides by incorporating mass linkers assembled using 4-aminomethyl benzoic acid derivatives. The mass linkers can be modified by incorporating one or two fluorine atoms to further space out the mass differences between the nucleotides. The structures of the newly designed biotinylated ddNTPs are shown in
The DNA sequencing fragments that carry a biotin at the 3′-end are made free from salts and other components in the sequencing reaction by capture with streptavidin-coated magnetic beads. Thereafter, the correctly terminated biotinylated DNA fragments are released and loaded onto the mass spectrometer. Results indicate that MS can produce high resolution of DNA-sequencing fragments, fast separation on microsecond time scales, and eliminate the compressions associated with gel electrophoresis.
Amplification products obtained by PCR with broad range 5′-UTR or polymerase 3D primer sets can be used as template. Sequencing permits discrimination between bona fide enteroviral amplification products and artifacts. Where analysis of the semi-divergent sequence region located toward the 3′-end of the 5′-UTR region is inadequate for speciation, targeting the more divergent VP3 and/or VP1 regions is preferred.
Background and Significance
The advent of SARS in 2003 poignantly demonstrated the urgency of establishing rapid, sensitive, specific, inexpensive tools for differential laboratory diagnosis of infectious diseases. Through unprecedented global collaborative efforts, the causative agent was rapidly implicated and characterized, facilitating development of serologic and molecular assays for infection, and containment of the outbreak. Nonetheless, as the northern hemisphere entered the winter season of 2004, the diagnosis of SARS still rested on clinical and epidemiological as well as laboratory criteria.
Methods for cloning nucleic acids of microbial pathogens directly from clinical specimens offer new opportunities to investigate microbial associations in diseases. The power of these methods is not only sensitivity and speed but also the potential to succeed where methods for pathogen identification through serology or cultivation may fail due to absence of specific reagents or fastidious requirements for agent replication.
Various methods are employed or proposed for cultivation-independent characterization of infectious agents. These can be broadly segregated into methods based on direct analysis of microbial nucleic acid sequences, direct analysis of microbial protein sequences, immunological systems for microbe detection, and host response profiling. Any comprehensive armamentarium should include most, if not all, of these tools. Nonetheless, classical methods for microbiology remain important. Indeed, the critical breakthrough during the SARS outbreak was the cultivation of the agent in tissue culture.
Real-time PCR methods have significantly changed diagnostic molecular microbiology by providing rapid, sensitive, specific tools for detecting and quantitating genetic targets. Because closed systems are employed, real-time PCR is less likely than nested PCR to be confounded by assay contamination due to inadvertent aerosol introduction of amplicon/positive control/cDNA templates that can accumulate in diagnostic laboratories. The specificity of real time PCR is both a strength and a limitation. Although the potential for false positive signal is low so is the utility of the method for screening to detect related but not identical genetic targets. Specificity in real-time PCR is provided by two primers (each approximately 20 matching nucleotides (nt) in length) combined with a specific reporter probe of about 27 nt. The constraints of achieving hybridization at all three sites may confound detection of diverse, rapidly evolving microbial genomes such as those of single-stranded RNA viruses. These constraints can be compensated in part by increasing numbers of primer sets accommodating various templates. However, because real-time PCR relies on fluorescent reporter dyes, the capacity for multiplexing is limited to the number of emission peaks that can be unequivocally separated. At present up to four dyes can be identified simultaneously. Although the repertoire may increase, it will unlikely to change dramatically.
To address the need for enhanced multiplex capacity in diagnostic molecular microbiology we have established a PCR platform based on mass tag reporters that are easily distinguished in MS as discrete signal peaks. Major advantages of the PCR/MS system include: (1) hybridization to only two sites is required (forward and reverse primer binding sites) vs real time PCR where an intermediate third oligonucleotide is used (probe binding site); this enhances flexibility in primer design; (2) tried and proven consensus PCR primers can be adapted to PCR/MS; this reduces the time and resources that must be invested to create new reagents and assay controls; (3) the large repertoire of tags allows highly multiplexed assays; additional tags can be easily synthesized to allow further complexity; and (4) sensitivity of real time PCR is maintained. We view PCR/MS as a tool with which to rapidly screen clinical materials for the presence of candidate pathogens. Thereafter, targeted secondary tests, including real time PCR, can be used to quantitate microbe burden and pursue epidemiologic studies.
Preliminary Data
We have developed bioinformatic tools to facilitate sequence alignments, motif identification, and primer design; established banks of viral strains, cDNA templates, and primers; and built relationships with collaborators in national and global public health laboratory networks that provide access to data, organisms, sera, and cDNAs that facilitate assay development and validation. Over the past two years we have integrated PCR and MS into a stable and sensitive digital assay platform similar in sensitivity and efficiency to real time PCR but with the advantages of simultaneous detection and discrimination of multiple targets. Using the 4 tags created for DNA sequencing we initially tested the method with flavivirus and bunyavirus targets as a proof of principle for an encephalitis project. The collaboration was later expanded to include two industrial partners: QIAGEN GmbH, a partner with a large validated library of proprietary photocleavable mass tags (Masscode™) and expertise in manufacture and commercial distribution, and Griffin Analytical Technologies, a partner actively engaged in design and fabrication of low cost portable MS instruments for field applications.
Selection of APCI LCMS Platform
Mass spectrometry is a rapid, sensitive method for detection of small molecules. With the development of Ionization techniques such as matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI), MS has become a indispensable tool in many areas of biomedical research. Although these ionization methods are suitable for the analysis of bioorganic molecules, such as peptides and proteins, improvements in both detection and sample preparation will be required before mass spectrometry can be used to directly detect long DNA fragments. A major confound in exploiting MS for genetic investigation has been that long DNA molecules are fragmented during the analytic process. The mass tag approach we have developed overcomes this limitation by detecting small stable mass tags that serve as signatures for specific DNA sequences rather than the DNA sequences themselves.
We have explored the kinetics of photocleavable primer conjugation. Ionization and detection of the photocleaved mass tags have been extensively characterized using atmospheric pressure chemical ionization (APCI) as the ionization source while using a single quadrupole mass spectrometer as the detector (Jingyue et al., Kim et al. 2003; Kokoris et al. 2000). Because buffer and inorganic salts impact ionization efficiency, performance in ESI was determined to be critically dependent upon sample preparation conditions. In MALDI, matrix must be added prior to sample introduction into the mass spectrometer, which is a time consuming step that requires costly sample spotting instrumentation. Similary, speed is often limited by the need to search for an ideal irradiation spot to obtain interpretable mass spectra.
In contrast, APCI is much more tolerant of residual inorganic salts (than ESI) and does not require mixing with matrix to prepare crystals on a target plate. Thus, mass tag solutions can be injected directly into the MS via a Liquid Chromatography (LC) delivery system. Since mass tags ionize well under APCI conditions and have small mass values (less that 800 amu), they are detected with high sensitivity (<5 femtomolar limit of detection) with the APCI-Quadrupole LCMS platform.
Methods for synthesis and APCI-MS analysis of mass tags coupled to DNA fragments are illustrated in
Upon nitration and reduction, the photoactive tags are produced and used to code for the identity of different primer pairs. An example for photocleavage and detection of four tags is shown in
Establishment of a PCR/MS Assay for Respiratory Pathogens
During the SARS 2003 Beijing outbreak we established a specific and sensitive real time PCR assay for SARS-CoV (Zhai et al, 2004). The assay was extended to allow simultaneous detection of SARS-CoV as well as human coronaviruses OC43 and 229E in light of recent data from China suggesting the potential for coinfection and increased morbidity (
To build a more comprehensive respiratory pathogen surveillance assay we adapted the human coronavirus primers to the PCR/MS platform, and added reagents required to detect other relevant microbes. Influenza A virus was included through a set of established primer sequences obtained through Georg Pauli (Robert Koch Institute, Germany; Schwaiger et al 2000). For the bacterial pathogen M. pneumoniae we also used unmodified primer sequences published for real time PCR (Welti et al 2003) to evaluate their use on the PCR/MS platform. Using a panel of mass tags developed by QIAGEN, experiments were performed demonstrating the feasibility of detecting several respiratory pathogens in a single multiplexed assay on the PCR/MS platform.
The current Masscode™ photocleavable mass tag repertoire comprises over 80 tags.
The presence of mass tags did not impair performance of primers in PCR and yielded clear signals for all 5 agents (
Establishment of a Platform for Portable MS
Griffin has developed a portable mass spectrometer that is roughly the size of a tower computer (including vacuum system), weighs less than 50 lbs, and consumes ˜150 W depending on operating conditions. This system has a mass range of 400 Da with unit mass resolution. It has been used to detect part-per-trillion level atmospheric constituents.
Experimental Design
Labeled amplification products are generated during PCR amplification with mass tagged primers. After isolation from non-incorporated primers by binding to silica in Qiagen 96-well or 384-well PCR purification modules, products are eluted into the injection module of the mass-spectrometer. The products traverse the path of a UV light source prior to entering the nebulizer, releasing photocleavable tags. (one each from the forward and reverse primer). Mass tags are then ionized. Analysis of the mass code spectrum defines the pathogen composition of the specimen.
A non-comprehensive list of target pathogens is listed in Tables 2 and 3. Forward and reverse primer pairs for pathogens listed in Table 2 are (reading from top to bottom starting with RSV-A and ending with M. Pneumoniae), SEQ ID NOS:1 and 2, 3 and 4, 9 and 10, 21 and 22, 23 and 24, 26 and 27, and 49 and 50.
Design and Synthesis of Primers
Primers are designed using the same approach as employed for the 7-plex assay. Available sequences are be extracted from GenBank. Conserved regions suitable for primer design are identified using standard software programs as well as custom software (patent application XYZ). Primer properties can be assessed by commercial primer selection software including OLIGO (Molecular Biology Insights), Primer Express (PE Applied Biosystems), and Primer Premiere (Premiere Biosoft International). Primers are evaluated for signal strength and specificity against a background of total human DNA.
Isolation and Cloning of Template Standards
Targeted genes can be cloned into the transcription vector pGEM-Teasy (Invitrogen) by conventional RT-PCR cloning methods. Quantitated plasmid standards are used in initial assay establishment. Thereafter, RNA transcripts generated by in vitro transcription, quantitated and diluted in a background of random human RNA (representing brain, liver, spleen, lung and placenta in equal proportions) are employed to establish sensitivity and specificity parameters of RT-PCR/MS assays. One representative isolate for each targeted pathogen/gene is used during initial establishment of the assay.
Inherent in the exquisite sensitivity of PCR is the risk of false positive results due to inadvertent introduction of synthetic templates such as those comprising positive control and calibration reagents, and so calibration reagents are preferred components of kits. Thus, to allow recognition of control vs authentic, natural amplification products, calibration reagents are modified by introducing a restriction enzyme cleavage site in between the primer binding sites through site directed mutagenesis. This approach has been employed in projects concerned with epidemiology of viral infection in various chronic diseases including Bornaviruses in neuropsychiatric disease (NIH/MH57467), measles virus in autism (CDC/American Academy of Pediatrics), and enteroviruses in type I diabetes mellitus (NIH/AI55466).
Multiplex Assay Using Cloned Template Standards
Initially, the performancance of individual primer sets with unmodified primers is tested. Amplification products in these single assays can be detected by gel electrophoresis. This strategy will not serve for multiplex assays because products of individual primer sets will be similar in size i.e. <300 bp. Thus, after confirmation of performance in single assays, mass tagged primers are generated for multiplex analyses. All assays are first optimized for PCR using serial dilutions of plasmid DNA, and then for RT-PCR using serial dilutions of synthetic transcripts. A multiplex assay is considered successful if it detects all target sequences at a sensitivity of 50 copies plasmid DNA per assay and 100 copies RNA per assay. Successful multiplex assay performance includes detection of all permutative combinations of two agents to ensure the feasibility of diagnosing simultaneous infection.
Optimizing Multiplex Assay Using Cell Culture Extracts
After establishing performance parameters with calibrated synthetic reagents, cell culture extracts of authentic pathogens are used. Performance of assays with RNA extracted using readily available commercial systems that do or do not include organic solvents (e.g, Tri-Reagent vs RNeasy) is assessed. A protocol disclosed here employs Tri-Reagent. Similarly, although Superscript reverse transcriptase (Invitrogen) and HotStart polymerase (QIAGEN) can be used, performance of ThermoScript RT (Invitrogen) at elevated temperature can be assessed, as are single-step RT-PCR systems like the Access Kit (Promega). To optimize efficiency where clinical material mass is limited and to reduce the complexity of sample preparation, both viral and bacterial agents can be identified using RT-PCR. Where an agent is characterized by substantive phylogenetic diversity, cell culture systems should include at least three divergent isolates of each pathogen
Sample Processing
Samples may be obtained by nasal swabs, sputum and lavage specimens will be spiked with culture material to optimize recovery methods for viral as well as bacterial agents.
Portable APCI MS Instruments to Support Multiplex PCR/MS Platform
The multiplex mass tag approach is well-suited to implementation on a miniaturized MS system, as the photocleavable mass tags are all relatively low in molecular weight (<500 Da.), and hence the constraints on the mass spectrometer in terms of mass range and mass resolution are not high. The technical challenge associated with this approach is the development of an atmospheric-pressure chemical ionization (APCI) source for use on a miniaturized MS to generate the mass tag ions. Such a source has been coupled with a miniaturized MS in an academic setting.
Detection of NIAD Category A, B, and C Priority Agents
Using the same approach as outlined for respiratory pathogen detection, a multiplex assay for detection of selected NIAD Category A, B, and C priority agents can be created (Table 3). Primers and PCR conditions for several agents are already established and can be adapted to the PCR/MS platform.
Background
Efficient laboratory diagnosis of infectious diseases is increasingly important to clinical management and public health. Methods for direct detection of nucleic acids of microbial pathogens in clinical specimens are rapid, sensitive and may succeed where fastidious requirements for agent replication confound cultivation. Nucleic acid amplification systems are indispensable tools in HIV and HCV diagnosis, and are increasingly applied to pathogen typing, surveillance, and diagnosis of acute infectious disease. Clinical syndromes are only infrequently specific for single pathogens; thus, assays for simultaneous consideration of multiple agents are needed. Current multiplex assays employ gel-based formats where products are distinguished by size, fluorescent reporter dyes that vary in color, or secondary enzyme hybridization assays. Gel-based assays are reported that detect 2-8 different targets with sensitivities of 2-100 pfu or <1-5 pfu, depending on whether amplification is carried out in a single or nested format, respectively (Ellis and Zambon 2002, Coiras et all. 2004). Fluorescence reporter systems achieve quantitative detection with sensitivity similar to nested amplification; however, their capacity to simultaneously query multiple targets is limited to the number of fluorescent emission peaks that can be unequivocally separated. At present up to four fluorescent reporter dyes are detected simultaneously (Vet et al. 1999, Verweij et al. 2004). Multiplex detection of up to 9 pathogens was achieved in hybridization enzyme systems; however, the method requires cumbersome post-amplification processing (Gröndahl et al. 1999).
To address the need for sensitive multiplex assays in diagnostic molecular microbiology we created a polymerase chain reaction (PCR) platform wherein microbial gene targets are coded by 64 distinct mass tags. Here we describe this system, mass tag PCR, and demonstrate its utility in differential diagnosis of respiratory tract infections.
Oligonucleotide primers for mass tag PCR were designed to detect the broadest number of members for a given pathogen species through efficient amplification of a 50-300 basepair product. In some instances we selected established primer sets; in others we employed a software program designed to cull sequence information from GenBank, perform multiple alignments, and maximize multiplex performance by selecting primers with uniform melting temperatures and minimal cross-hybridization potential. Primers, synthesized with a 5′ C6-spacer and aminohexyl modification, were covalently conjugated via a photocleavable linkage to small molecular weight tags (Kokoris et al. 2000) to encode their respective microbial gene targets. Forward and reverse primers were labeled with differently sized tags to produce a dual code for each target that facilitates assessment of signal specificity.
Microbial gene target standards for sensitivity and specificity assessment were cloned by PCR using cDNA template obtained by reverse transcription of extracts from infected cultured cells or by assembly of overlapping synthetic polynucleotides. Cloned standards representing genetic sequence of the targeted microbial pathogens were diluted in 12.5 ug/ml human placenta DNA (Sigma, St. Louis, Mo., USA) and subjected to multiplex PCR amplification using the following cycling protocol: 9×C for X sec., 55 C for X sec., 72 C for X sec.; 50 cycles, MJ PTC200 (MJ Research, Waltham, Mass., USA). Amplification products were purified using QIAquick 96 PCR purification cartridges (Qiagen, Hilden, Germany) with modified binding and wash buffers (RECIPES). Mass tags of the amplified products were analyzed after ultraviolet photolysis and positive-mode atmospheric pressure chemical ionization (APCI) by single quadrapole mass spectrometry.
We next analyzed samples from individuals with respiratory infection using a larger panel comprising 30 gene targets (26 pathogens). Mass Tag PCR correctly identified infection with respiratory syncitial, human parainfluenza, SARS corona, adeno, entero, metapneumo and influenza viruses (Table 4 and
Our results indicate that mass tag PCR is a useful method for molecular characterization of microflora. Sensitivity is similar to real time PCR assays but with the advantage of allowing simultaneous screening for several candidate pathogens. Potential applications include differential diagnosis of infectious diseases, blood product surveillance, forensic microbiology, and biodefense.
Multiplex PCR
Conventional multiplex PCR assays are established, however, none allow sensitive detection of more than 10 genetic targets. The most sensitive of these assays, real time PCR, is limited to four fluorescent reporter dyes. Gel based systems are cumbersome and limited to visual distinction of products that differ by 20 bp; multiplexing is restricted to the number of products that can be distinguished at 20 bp intervals within the range of 100 to 250 bp (amplification efficiency decreases with larger products); nesting or Southern hybridization is required for high sensitivity. A 9-plex assay has been achieved using hybridization capture enzyme assay.
Disclosed here are panels of nucleic acid sequences to be used in assays for the detection of infectious agents. The sequences include primers for polymerase chain reaction, enzyme sites for initiating isothermal amplification, hybridization selection of nucleic acid targets, as well as templates to serve as controls for validation of these assays. This example focuses on the use of these panels for multiplex mass tag PCR applications. Nucleic acid databases were queried to identify regions of sequence conservation within viral and bacterial taxa wherein primers could be designed that met the following critera: (i) the presence of motifs required to create specific or low degeneracy PCR primers that targeted all members of a microbial group (or subgroup); (ii) Tm of 59-61 C; (iii) GC content of 48-60%; (iv) length of 18-24 bp; (v) no more than three consecutive identical bases; (vi) 3 or more G and/or C residues in the 5′-hexamer; (vii) less than 3 G and/or C residues in the 3′-pentamer; (vii) no propensity for secondary structure (stem-loop) formation; (viii) no inter-primer complementarity that could predispose to primer-dimer formation; (ix) amplification of an 80-250 bp region with no or little secondary structure at 59-61 C. Primers meeting these criteria were then evaluated empirically for equal performance in context of the respective multiplex panel. In the event that no ideal primer candidates could be identified, primers that did not meet one or more of these criteria were synthesized and evaluated for appropriate performance. Those that yielded 80-250 bp amplification products, had Tm of 59-61 C, and showed no primer-dimer artifacts were selected for inclusion into panels.
As a proof-of-principle we designed a panel of primers for detection of 31 target sequences of respiratory pathogens (25-plex respiratory panel) and demonstrated successful detection of all potential targets in a 25-plex PCR reaction. Detection of amplification products was achieved through use of the MASSCODE® technology. Individual primers were conjugated with a unique masscode tag through a photocleavable linkage. Photocleavage of the masscode tag from the purified PCR product and mass spectrometric analysis identifies the amplified target through the two molecular weights assigned to the forward and reverse primer. Primer panels focus on groups of infectious pathogens that are related to differential diagnosis of respiratory disease, encephalitis, or hemorrhagic fevers; screening of blood products; biodefense; food safety; environmental contamination; or forensics.
Background and Significance
The advent of SARS in 2003 poignantly demonstrated the urgency of establishing rapid, sensitive, specific, inexpensive tools for differential laboratory diagnosis of infectious diseases. Through unprecedented global collaborative efforts, the causative agent was rapidly implicated and characterized, facilitating development of serologic and molecular assays for infection, and containment of the outbreak. Nonetheless, as the northern hemisphere entered the winter season of 2004, the diagnosis of SARS still rests on clinical and epidemiological as well as laboratory criteria. The WHO SARS International Reference and Verification Laboratory Network met on Oct. 22, 2003 to review the status of laboratory diagnostics in acute severe pulmonary disease. Quality assurance testing indicated that false positive SARS CoV PCR results were infrequent in network labs. However, participants registered concern that current assays did not allow simultaneous detection of a wide range of pathogens that could aggravate disease or themselves result in clinical presentations similar to SARS.
Methods for cloning nucleic acids of microbial pathogens directly from clinical specimens offer new opportunities to investigate microbial associations in diseases. The power of these methods is not only sensitivity and speed but also the potential to succeed where methods for pathogen identification through serology or cultivation may fail due to absence of specific reagents or fastidious requirements for agent replication.
Various methods are employed or proposed for cultivation-independent characterization of infectious agents. These can be broadly segregated into methods based on direct analysis of microbial nucleic acid sequences, direct analysis of microbial protein sequences, immunological systems for microbe detection, and host response profiling. Any comprehensive armamentarium should include most, if not all, of these tools. Nonetheless, classical methods for microbiology remain important. Indeed, the critical breakthrough during the SARS outbreak was the cultivation of the agent in tissue culture.
Real-time PCR methods have significantly changed diagnostic molecular microbiology by providing rapid, sensitive, specific tools for detecting and quantitating genetic targets. Because closed systems are employed, real-time PCR is less likely than nested PCR to be confounded by assay contamination due to inadvertent aerosol introduction of amplicon/positive control/cDNA templates that can accumulate in diagnostic laboratories. The specificity of real time PCR is both, a strength and a limitation. Although the potential for false positive signal is low so is the utility of the method for screening to detect related but not identical genetic targets. Specificity in real-time PCR is provided by two primers (each approximately 20 matching nucleotides (nt) in length) combined with a specific reporter probe of about 27 nt. The constraints of achieving hybridization at all three sites may confound detection of diverse, rapidly evolving microbial genomes such as those of single-stranded RNA viruses. These constraints can be compensated in part by increasing numbers of primer sets accommodating various templates. However, because real-time PCR relies on fluorescent reporter dyes, the capacity for multiplexing is limited to the number of emission peaks that can be unequivocally separated. At present up to four dyes can be identified simultaneously. Although the repertoire may increase, it will unlikely to change dramatically.
To address the need for enhanced multiplex capacity in diagnostic molecular microbiology we have established a PCR platform based on mass tag reporters that are easily distinguished in MS as discrete signal peaks. Major advantages of the PCR/MS system include: (1) hybridization to only two sites is required (forward and reverse primer binding sites) vs real time PCR where an intermediate third oligonucleotide is used (probe binding site); this enhances flexibility in primer design; (2) tried and proven consensus PCR primers can be adapted to PCR/MS; this reduces the time and resources that must be invested to create new reagents and assay controls; (3) the current repertoire of 60 tags allows highly multiplexed assays; additional tags can be easily synthesized to allow further complexity; and (4) sensitivity of real time PCR is maintained. A limitation of PCR/MS is that it is unlikely to provide more than a semi-quantitative index of microbe burden. Thus, we view PCR/MS as a tool with which to rapidly screen clinical materials for the presence of candidate pathogens. Thereafter, targeted secondary tests, including real time PCR, should be used (to quantitate microbe burden and pursue epidemiologic studies.
Selection of APCI LCMS Platform
Mass spectrometry is a rapid, sensitive method for detection of small molecules. With the development of Ionization techniques such as matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI), MS has become a indispensable tool in many areas of biomedical research. Although these ionization methods are suitable for the analysis of bioorganic molecules, such as peptides and proteins, improvements in both detection and sample preparation will be required before mass spectrometry can be used to directly detect long DNA fragments. A major confound in exploiting MS for genetic investigation has been that long DNA molecules are fragmented during the analytic process. The mass tag approach we have developed overcomes this limitation by detecting small stable mass tags that serve as signatures for specific DNA sequences rather than the DNA sequences themselves.
Ionization and detection of the photocleaved mass tags have been extensively characterized using atmospheric pressure chemical ionization (APCI) as the ionization source while using a single quadrupole mass spectrometer as the detector (Jingyue et al., Kim et al. 2003; Kokoris et al. 2000). Because buffer and inorganic salts impact ionization efficiency, performance in ESI was determined to be critically dependent upon sample preparation conditions. In MALDI, matrix must be added prior to sample introduction into the mass spectrometer, which is a time consuming step that requires costly sample spotting instrumentation. Similarly, speed is often limited by the need to search for an ideal irradiation spot to obtain interpretable mass spectra. In contrast, APCI is much more tolerant of residual inorganic salts (than ESI) and does not require mixing with matrix to prepare crystals on a target plate. Thus, mass tag solutions can be injected directly into the MS via a Liquid Chromatography (LC) delivery system. Since mass tags ionize well under APCI conditions and have small mass values (less that 800 amu), they are detected with high sensitivity (<5 femtomolar limit of detection) with the APCI-Quadrupole LCMS platform.
Methods for synthesis and APCI-MS analysis of mass tags coupled to DNA fragments are illustrated in
Upon nitration and reduction, the photoactive tags are produced and used to code for the identity of different primer pairs. An example for photocleavage and detection of four tags is shown in
The mechanism for release of these tags from DNA is shown in
This result indicates that the 4 compounds designed as mass tags are stable and produce discrete high-resolution digital data in an APCI mass spectrometer. In the research plan described below, the unique m/z from each mass tag will translate to the identity of a viral sequence. Qiagen has developed a large library of more than 80 proprietary masscode tags (Kokoris et al. 2000). Examples are shown in
Establishment of a PCR/MS Assay for Respiratory Pathogens
During the SARS 2003 Beijing outbreak we established a specific and sensitive real time PCR assay for SARS-CoV (Zhai et al, 2004). The assay was extended to allow simultaneous detection of SARS-CoV as well as human coronaviruses OC43 and 229E in light of recent data from China suggesting the potential for coinfection and increased morbidity (
To build a more comprehensive respiratory pathogen surveillance assay we adapted the human coronavirus primers to the PCR/MS platform, and added reagents required to detect other relevant microbes. Influenza A virus was included through a set of established primer sequences obtained through Georg Pauli (Robert Koch Institute, Germany; Schwaiger et al 2000). For the bacterial pathogen M. pneumoniae we also used unmodified primer sequences published for real time PCR (Welti et al 2003) to evaluate their use on the PCR/MS platform. Using a panel of mass tags developed by QIAGEN, pilot experiments were performed, demonstrating the feasibility of detecting several respiratory pathogens in a single multiplexed assay on the PCR/MS platform.
Subsequent to the 1999 West Nile Virus (WNV) outbreak in the U.S. we also built a real time PCR assay for differential diagnosis of flaviviruses WNV and St. Louis encephalitis virus—see
The current Masscode photocleavable mass tag repertoire comprises over 80 tags.
Establishment of a Platform for Portable MS
Griffin has developed a portable mass spectrometer that is roughly the size of a tower computer (including vacuum system), weighs less than 50 lbs, and consumes ˜150 W depending on operating conditions. This system has a mass range of 400 Da with unit mass resolution. It has been used to detect part-per-trillion level atmospheric constituents. Included below is a representative spectrum of methyl salicylate collected on a miniature cylindrical ion trap mass analyzer coupled to a corona discharge ionization source (data collected in Prof. R. G. Cooks research laboratory at Purdue University). This data demonstrates the feasibility of using this type of instrumentation to detect the mass tags of interest as well as the specificity of the ionization source.
Griffin has developed a mass spectrometer for field transportable use. Power consumption, weight, size, and ease of use have been focus design points in the development of this instrument. It has not been designed specifically for interface to an atmospheric pressure ionization (API) source like the one proposed here for pathogen surveillance and discovery. Thus, our focus in this proposal is directed toward the integration of an atmospheric pressure chemical ionization (APCI) source and the required vacuum, engineering, and software considerations associated with this integration.
Experimental Design
A cartoon of the assay procedure is shown in
The repertoire of potential pathogens to be targeted during this project is listed in Table 13. Forward and reverse primer pairs for pathogens listed in Table 13 are (reading from top to bottom starting with RSV-A and ending with M. Pneumoniae), SEQ ID NOS:1 and 2, 3 and 4, 9 and 10, 21 and 22, 23 and 24, 26 and 27, and 49 and 50.
Design and Synthesize Primers
Missing primers will be designed using the same approach as employed for the 7-plex assay. Available sequences will be extracted from GenBank. Conserved regions suitable for primer design will be identified using standard software programs as well as custom software (patent application XYZ). Primer properties will be assessed by commercial primer selection software including OLIGO (Molecular Biology Insights), Primer Express (PE Applied Biosystems), and Primer Premiere (Premiere Biosoft International). Non-tagged primers will be synthesized, and performance assessed using cloned target sequences as described in preliminary data. Primers will be evaluated for signal strength and specificity against a background of total human DNA. Currently, 80% of primers perform as predicted by our algorithms. Thus, to minimize delay we typically synthesize multiple primer sets for similar genetic targets and evaluate their performance in parallel.
Inherent in the exquisite sensitivity of PCR is the risk of false positive results due to inadvertent introduction of synthetic templates such as those comprising positive control and calibration reagents. Calibration reagents will be components of kits distributed to network laboratories and customers. Thus, to allow recognition of control vs authentic, natural amplification products, we will modify calibration reagents by introducing a restriction enzyme cleavage site in between the primer binding sites through site directed mutagenesis. We have used this approach in projects concerned with epidemiology of viral infection in various chronic diseases including Bornaviruses in neuropsychiatric disease (NIH/MH57467), measles virus in autism (CDC/American Academy of Pediatrics), and enteroviruses in type I diabetes mellitus (NIH/AI55466).
Establish Multiplex Assay Using Cloned Template Standards
Before committing resources to generating mass tagged primers we will test the performance of individual primer sets with unmodified primers. Amplification products in these single assays will be detected by gel electrophoresis. This strategy will not serve for multiplex assays because products of individual primer sets will be similar in size i.e., all will be <300 bp. Although individual products in multiplex assays could be resolved by sequence analysis our experience suggests it will be more cost effective to proceed directly to PCR/MS analysis. Thus, after-performance is confirmed in single assays we will generate mass tagged primers for multiplex analyses. All assays will be optimized first for PCR using serial dilutions of plasmid DNA, and then for RT-PCR using serial dilutions of synthetic transcripts. A multiplex assay will be considered successful if it detects all target sequences at a sensitivity of 50 copies plasmid DNA per assay and 100 copies RNA per assay. Successful multiplex assay performance will also include detection of all permutative combinations of two agents to ensure the feasibility of diagnosing simultaneous infection.
Optimize Multiplex Assay Using Cell Culture Extracts
After establishing performance parameters with calibrated synthetic reagents, cell culture extracts of authentic pathogens will be used. We will recommend specific kits for nucleic acid extraction and RT-PCR. Nonetheless, we recognize that some investigators may choose to use other reagents. Thus, we will assess performance of assays with RNA extracted using readily available commercial systems that do or do not include organic solvents (e.g, Tri-Reagent vs RNeasy). Our current protocol employs Tri-Reagent. Similarly, although we use Superscript reverse transcriptase (Invitrogen) and HotStart polymerase (QIAGEN), we will also assess the performance of ThermoScript RT (Invitrogen) at elevated temperature, and of single-step RT-PCR systems like the Access Kit (Promega). To optimize efficiency where clinical material mass is limited and to reduce the complexity of sample preparation, both viral and bacterial agents will be identified using RT-PCR. In the event network collaborators agree an agent is characterized by substantive phylogenetic diversity, cell culture systems will include at least three divergent isolates of each pathogen. Nasal swabs, sputum and lavage specimens will be spiked with culture material to optimize recovery methods for viral as well as bacterial agents. Assays are validated using banked specimens from naturally infected humans, and naturally infected animals.
Primer Design and Synthesis, Template Design and Synthesis
Respiratory Panel includes 27 gene targets with validated primer sets as shown below in Table 5. Forward and reverse primer pairs (SEQ ID NOs:1-54) are given for each pathogen (reading from top to bottom starting with RSV-A and ending with C. Pneumoniae). For example, forward primer for RSV-A is SEQ ID NO:1, reverse primer for RSV-A is SEQ ID NO:2. Forward primer for RSV-B is SEQ ID NO:3, reverse primer for RSV-B is SEQ ID NO:4, etcetera.
Table 6, NIAID Priority Agent Panel.
Assays have been designed using 4 primer sets and their cognate synthetic Rift Valley Fever, Crimean Congo Hemorrhagic Fever, Ebola Zaire and Marburg virus templates created via PCR using overlapping polynucleotides, as shown in Table 6. Forward and reverse primer pairs (SEQ ID NOs:55-62) are given for four of the listed pathogens (reading from top to bottom starting with Rift Valley Fever virus and ending with Marburg virus). For example, forward primer for Rift Valley Fever virus is SEQ ID NO:55, reverse primer for Rift Valley Fever virus is SEQ ID NO:56. Forward primer for CCHF virus is SEQ ID NO:57, reverse primer for CCHF virus is SEQ ID NO:58, etcetera.
Encephalitis Agent Panel
Table 7 shows primer sets for encephalitis-inducing agents. Forward and reverse primer pairs (SEQ ID NOs:63-96) are given for each pathogen (reading from top to bottom starting with West Nile virus and ending with Enterovirus). For example, forward primer for West Nile virus is SEQ ID NO:63, reverse primer for West Nile virus is SEQ ID NO:64. Forward primer for St. Louis Encephalitis virus is SEQ ID NO:65, reverse primer for St. Louis Encephalitis virus is SEQ ID NO:66, etcetera.
Improvements in Multiplexing
Initially, multiplex detection of 7 respiratory pathogen targets at 500 copy sensitivity: RSV group A, RSV group B, Influenza A, HCoV-SARS, HCoV-229E, HCoV-OC43, and M. pneumoniae was determined. Subsequently, sensitivity was improved. Detection at 100 copy sensitivity has been confirmed for 18 respiratory pathogen targets in a 20-plex assay (Table 8). Two of 20 targets, the influenza A M gene and influenza H1 gene, were detected at 500 copies. This typically corresponds in our laboratory to <0.001 TCID50 per assay, a threshold comparable to many useful microbiological assays.
Clinical Samples
Although assays of synthetic targets were optimized in a complex background of normal tissue nucleic acids, analysis of clinical materials was performed. Banked clinical respiratory specimens were obtained from Cinnia Huang of the Wadsworth Laboratory of the New York State Department of Health and Pilar Perez-Brena of the National Center for Microbiology of Spain. Organisms included: metapneumovirus (n=3), RSV-B (n=3), RSV-A (n=2), adenovirus (n=2), HPIV-1 (n=1), HPIV-3 (n=2), HPIV-4 (n=2), enterovirus (n=2), SARS-CoV (n=4), influenza A (n=2). Six representative results are shown in
Pathogens
Tables 9-12 show a non-comprehenisve list of various target pathogens and corresponding primer sequences. In Table 10, the forward and reverse primer pairs for Cytomegalovirus, SEQ ID NOS: 87 and 88; for HPIV-4A, SEQ ID NOS: 37 and 38; for HPIV-4B, SEQ ID NOS: 39 and 40; for Measles, SEQ ID NOS: 91 and 92; for Varicella Zoster virus, SEQ ID NOS: 89 and 90; for HIV-1, SEQ ID NOS: 69 and 70; for HIV-2, SEQ ID NOS: 71 and 72; for S. Pneumoniae, SEQ ID NOS: 100 and 101; for Haemophilus Influenzae, SEQ ID NOS: 77 and 78; for Herpes Simplex, SEQ ID NOS: 67 and 68; for MV Canadian isolates, SEQ ID NOS: 29 and 30; for Adenovirus 2 A/B 505/630, SEQ ID NOS: 93 and 94; for Enterovirus A/B 702/495, SEQ ID NOS: 95 and 96; and forward primers for Enterovirus A/B 702/495, SEQ ID NOS: 98 and 99.
Efficient laboratory diagnosis of infectious diseases is increasingly important to clinical management and public health. Methods to directly detect nucleic acids of microbial pathogens in clinical specimens are rapid, sensitive, and may succeed when culturing the organism fails. Clinical syndromes are infrequently specific for single pathogens; thus, assays are needed that allow multiple agents to be simultaneously considered. Current multiplex assays employ gel-based formats in which products are distinguished by size, fluorescent reporter dyes that vary in color, or secondary enzyme hybridization assays. Gel-based assays are reported that detect 2-8 different targets with sensitivities of 2-100 PFU or less than 1-5 PFU, depending on whether amplification is carried out in a single or nested format, respectively (1-4). Fluorescence reporter systems achieve quantitative detection with sensitivity similar to that of nested amplification; however, their capacity to simultaneously query multiple targets is limited to the number of fluorescent emission peaks that can be unequivocally resolved. At present, up to 4 fluorescent reporter dyes can be detected simultaneously (5,6). Multiplex detection of up to 9 pathogens has been achieved in hybridization enzyme systems; however, the method requires cumbersome postamplification processing (7).
Experimental Results
To address the need for sensitive multiplex assays in diagnostic molecular microbiology, we created a polymerase chain reaction (PCR) platform in which microbial gene targets are coded by a library of 64 distinct Masscode tags (Qiagen Masscode technology, Qiagen, Hilden, Germany). A schematic representation of this approach is shown in
*NA, not assessed;
RSV, respiratory syncytial virus;
CoV, coronavirus;
SARS, severe acute respiratory syndrome;
HPIV, human parainfluenza virus.
The sensitivity of Mass Tag PCR to detect live virus was tested by using RNA extracted from serial dilutions of titered stocks of coronaviruses (severe acute respiratory syndrome [SARS] and OC43) and parainfluenzaviruses (HPIV 2 and 3). A 100-μL volume of each dilution was analyzed. RNA extracted from a 1-TCID50/mL dilution, representing 0.025 TCID50 per PCR reaction, was consistently positive in Mass Tag PCR. RNA extracted from banked sputum, nasal swabs, and pulmonary washes of persons with respiratory infection was tested by using an assay panel comprising 30 gene targets that represented 22 respiratory pathogens. Infection in each of these persons had been previously diagnosed through virus isolation, conventional nested RT-PCR, or both. Reverse transcription was performed using random hexamers, and Mass Tag PCR results were consistent in all cases with the established diagnosis. Infections with respiratory syncytial virus, human parainfluenza virus, SARS coronavirus, adenovirus, enterovirus, metapneumovirus, and influenza virus were correctly identified (Table 16 and
*RSV, respiratory syncytial virus;
HPIV, human parainfluenza virus;
CoV, coronavirus;
SARS, severe acute respiratory syndrome.
†No. positive and consistent with previous diagnosis/number tested (with respective previous diagnosis).
A panel comprising gene targets representing 17 pathogens related to central nervous system infectious disease (influenza A virus matrix gene; influenza B virus; human coronaviruses 229E, OC43, and SARS; enterovirus; adenovirus; human herpesvirus-1 and -3; West Nile virus; St. Louis encephalitis virus; measles virus; HIV-1 and -2; and Streptococcus pneumoniae, Haemophilus influenzae, and Nesseria meningitidis) was applied to RNA obtained from banked samples of cerebrospinal fluid and brain tissue that had been previously characterized by conventional diagnostic RT-PCR. Two of 3 cases of West Nile virus encephalitis were correctly identified. Eleven of 12 cases of enteroviral meningitis were detected representing serotypes CV-B2, CV-B3, CV-B5, E-6, E-11, E-13, E-18, and E-30 (data not shown).
Our results indicate that Mass Tag PCR is a sensitive and specific tool for molecular characterization of microflora. The advantage of Mass Tag PCR is its capacity for multiplex analysis. Although the use of degenerate primers (e.g., enteroviruses and adenoviruses, and Table 16) may reduce sensitivity, the limit of multiplexing to detect specific targets will likely be defined by the maximal primer concentration that can be accommodated in a PCR mix. Analysis requires the purification of product from unincorporated primers and mass spectroscopy. Although these steps are now performed manually, and mass spectrometers are not yet widely distributed in clinical laboratories, the increasing popularity of mass spectrometry in biomedical sciences and the advent of smaller, lower-cost instruments could facilitate wider use additional pathogen panels, our continuing work is focused on optimizing multiplexing, sensitivity, and throughput. Potential applications include differential diagnosis of infectious diseases, blood product surveillance, forensic microbiology, and biodefense.
This application claims benefit of U.S. Provisional Application No. 60/566,967, filed Apr. 29, 2004, the contents of which are hereby incorporated by reference.
The invention disclosed herein was made with Government support under grant no. AI51292 from the National Institutes of Health. Accordingly, the U.S. Government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60566967 | Apr 2004 | US |
