WO2004053076A2 - Methods for rapid detection and identification of bioagents for environmental and product testing - Google Patents
Methods for rapid detection and identification of bioagents for environmental and product testing Download PDFInfo
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- WO2004053076A2 WO2004053076A2 PCT/US2003/038757 US0338757W WO2004053076A2 WO 2004053076 A2 WO2004053076 A2 WO 2004053076A2 US 0338757 W US0338757 W US 0338757W WO 2004053076 A2 WO2004053076 A2 WO 2004053076A2
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- C—CHEMISTRY; METALLURGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Definitions
- This invention relates to the field of environmental and product testing.
- the invention provides rapid detection and identification of bioagents in environmental and product samples.
- Microbial contamination of waters can lead to numerous serious issues. Bacterial contamination can cause adverse economic impacts for the affected areas by closing popular recreational areas for extended periods of time. This is particularly relevant in the heavily visited recreational waters and the swimming beaches in urban centers. In addition, bacterial contamination can cause severe illnesses such as gastrointestinal disorders. Microbial contaminants may not be limited to surface or recreational waters. In fact, a far more costly migration of these microbial contaminants is their movement to groundwater supplies which are used for drinking water.
- the cysts of Cryptosporidium are of increasing importance because of their presence in water supplies.
- four spindle-shaped motile sporozooites burst from the cyst to infect gut epithelial cells and continue their life cycle.
- Entamoeba histolytica another water- borne pathogen, can cause diarrhea or a more serious invasive liver abscess.
- these amebae are cytotoxic.
- Giardia is found in contaminated rivers and lakes and is also be contracted via contaminated foods. There is some evidence that a heavy infection of attached Giardia physically blocks the important transport of nutrients across the epithelium (see, for example, www.cellsalive.com/parasit.htm).
- E. coli can cause severe, even letlial illness.
- a multi-state outbreak of over 500 cases of E. coli O157:H7 infections was associated with a restaurant chain (www.dhss.state.mo.us/Mo ⁇ pi/moepil61.pdf).
- Listeria monocytogenes Associated with raw milk, soft cheeses, and raw meats, L.
- NLVs Norwalk-like viruses
- this assay can take anywhere from 3 to 6 days. During this time, the food products might decay beyond the selling point. Consumers might contract food poisoning from the products, putting them at risk for sickness or death. It is therefore preferable to have an assay that can locate and identify the offending microbes in a timeframe that is measured in hours, not days.
- the assay must also be very sensitive because the tested samples might have no more (and possibly less) than one cell per milliliter or gram of starting material.
- Listeria infections can start from as few as 10 cells. As with most traditional microbiological tests, this requires some form of microbe culture enrichment using a growth medium, but it is critical that this step not take too much time (see, for example, pubs . acs . org/subscribe/j ournals/tcaw/ 12/i03/pdf/303 willis .pdf) .
- Bacteria and their enzymes, along with some fungi and critical nutrient additives are cost effective agents for in-situ remediation (otherwise known as bioremediation) of hazardous wastes and subsurface pollution in soils, sediments and wastewaters.
- bioremediation also known as bioremediation
- the ability of each bacterial strain to degrade toxic waste depends on the nature of each contaminant. Since most sites are typically comprised of multiple pollutant types, the most effective approach to bioremediation is to use a mixture of bacterial species/strains, each specific to the degradation of one or more types of contaminants. It is critical to monitor the composition of the indigenous and added bacterial consortium in order to evaluate the activity level of the bacteria, and to permit modifications of the nutrients and other conditions for optimizing the bioremediation process.
- bioremediation site it is desirable to return a bioremediation site to its natural state following the bioremediation process.
- monitoring of levels of bioremediating bacteria becomes necessary in assessment of the return of the site to the natural state.
- Fungal bioremediation is also possible.
- This technology utilizes white-rot fungi to clean up a wide spectrum of soil pollutants, such as wood preservatives, polycyclic aromatic hydrocarbons, organochlorines, polychlorinated biphyenyls, dyes, pesticides, fungicides, herbicides, and others. Rapid throughput methods for detection and identification of the members of the indigenous and added bacterial and/or fungal consortium would greatly facilitate characterization of such bioremediative processes.
- Stachybotrys is a species of mold which has earned the title "toxic black mold,” as it is one of the most lethal, yet common forms. Stachybotrys can become airborne and cause serious respiratory difficulties, memory and hearing loss, hemorrhaging, dizziness and sometimes death. Prolonged exposure to this strain can impair memory.
- Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. Low-resolution MS may be unreliable when used to detect some known agents, if their spectral lines are sufficiently weak or sufficiently close to those from other living organisms in the sample. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to detect a particular organism. Antibodies face more severe diversity limitations than arrays. If antibodies are designed against highly conserved targets to increase diversity, the false alarm problem will dominate, again because threat organisms are very similar to benign ones.
- Antibodies are only capable of detecting known agents in relatively uncluttered environments.
- Several groups have described detection of PCR products using high resolution electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry (ESI-FT- ICR MS). Accurate measurement of exact mass combined with knowledge of the number of at least one nucleotide allowed calculation of the total base composition for PCR duplex products of approximately 100 base pairs.
- ESI-FT- ICR MS electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry
- Electrospray ionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MS may be used to determine the mass of double-stranded, 500 base-pair PCR products via the average molecular mass (Hurst et al, Rapid Commun. Mass Spec. 1996, 10, 377-382).
- MALDI-TOF matrix-assisted laser desorption ionization- time of flight
- U.S. Patent No. 5,849,492 describes a method for retrieval of phylogenetically informative DNA sequences which comprise searching for a highly divergent segment of genomic DNA surrounded by two highly conserved segments, designing the universal primers for PCR amplification of the highly divergent region, amplifying the genomic DNA by PCR technique using universal primers, and then sequencing the gene to determine the identity of the organism.
- U.S. Patent No. 5,965,363 discloses methods for screening nucleic acids for polymorphisms by analyzing amplified target nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods.
- WO 99/14375 describes methods, PCR primers and kits for use in analyzing preselected DNA tandem nucleotide repeat alleles by mass spectrometry.
- WO 98/12355 discloses methods of determining the mass of a target nucleic acid by mass spectrometric analysis, by cleaving the target nucleic acid to reduce its length, making the target single-stranded and using MS to determine the mass of the single-stranded shortened target. Also disclosed are methods of preparing a double-stranded target nucleic acid for MS analysis comprising amplification of the target nucleic acid, binding one of the strands to a solid support, releasing the second strand and then releasing the first strand which is then analyzed by MS. Kits for target nucleic acid preparation are also provided.
- PCT WO97/33000 discloses methods for detecting mutations in a target nucleic acid by nonrandomly fragmenting the target into a set of single-stranded nonrandom length fragments and determining their masses by MS.
- U.S. Patent No. 5,605,798 describes a fast and highly accurate mass spectrometer-based process for detecting the presence of a particular nucleic acid in a biological sample for diagnostic purposes.
- WO 98/21066 describes processes for determining the sequence of a particular target nucleic acid by mass spectrometry.
- Processes for detecting a target nucleic acid present in a biological sample by PCR amplification and mass spectrometry detection are disclosed, as are methods for detecting a target nucleic acid in a sample by amplifying the target with primers that contain restriction sites and tags, extending and cleaving the amplified nucleic acid, and detecting the presence of extended product, wherein the presence of a DNA fragment of a mass different from wild-type is indicative of a mutation.
- Methods of sequencing a nucleic acid via mass spectrometry methods are also described.
- 5,547,835 describe methods of sequencing nucleic acids using mass spectrometry.
- U.S. Patent Nos. 5,622,824, 5,872,003 and 5,691,141 describe methods, systems and kits for exonuclease-mediated mass spectrometric sequencing.
- the present invention is directed to methods of identifying a bioagent in a sample by determining a first molecular mass of a first amplification product of a first bioagent identifying amplicon from the sample and comparing the first molecular mass to a second molecular mass of a second bioagent identifying amplicon wherein both first and second bioagent identifying amplicons are correlative.
- These methods are applicable to environmental samples including, for example, air samples, water samples, soil samples, surface swab samples and samples from a building or a container.
- product samples including, for example, foodstuff and cosmetic samples.
- the present invention is also directed to methods of monitoring a bioremediation process by identifying bioagents in a sample by determining a first molecular mass of a first amplification product of a first bioagent identifying amplicon from the sample and comparing the first molecular mass to a second molecular mass of a second bioagent identifying amplicon wherein both first and second bioagent identifying amplicons are correlative.
- Figures 1A-1H and Figure 2 are consensus diagrams that show examples of conserved regions from 16S rRNA (Fig. 1A-1, 1A-2, 1A-3, 1A-4, and 1A-5), 23S rRNA (3'-half, Fig. IB, 1C, and ID; 5'-half, Fig. 1E-F), 23S rRNA Domain I (Fig. 1G), 23S rRNA Domain IV (Fig. 1H) and 16S rRNA Domain III (Fig. 2) which are suitable for use in the present invention.
- Lines with arrows are examples of regions to which intelligent primer pairs for PCR are designed. The label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram.
- the label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram.
- the nucleotide sequence of the 16S rRNA consensus sequence is SEQ ID NO: 3 and the nucleotide sequence of the 23 S rRNA consensus sequence is SEQ ID NO:4.
- Figure 2 shows a typical primer amplified region from the 16S rRNA Domain III shown in Figure 1A-1.
- Figure 3 is a schematic diagram showing conserved regions in RNase P. Bases in capital letters are greater than 90% conserved; bases in lower case letters are 80-90% conserved; filled circles designate bases which are 70-80% conserved; and open circles designate bases that are less than 70% conserved.
- Figure 4 is a schematic diagram of base composition signature determination using nucleotide analog "tags" to determine base composition signatures.
- Figure 5 shows the deconvoluted mass spectra of a Bacillus anthracis region with and without the mass tag phosphorothioate A (A*). The two spectra differ in that the measured molecular weight of the mass tag-containing sequence is greater than the unmodified sequence.
- Figure 6 shows base composition signature (BCS) spectra from PCR products from Staphylococcus aureus (S. aureus 16S_1337F) and Bacillus anthracis (B. anthr. 16S_1337F), amplified using the same primers.
- the two strands differ by only two (AT ⁇ >CG) substitutions and are clearly distinguished on the basis of their BCS.
- Figure 7 shows that a single difference between two sequences (A14 in B. anthracis vs. A15 in B. cereus) can be easily detected using ESI-TOF mass spectrometry.
- Figure 8 is an ESI-TOF of Bacillus anthracis spore coat protein sspE 56mer plus calibrant. The signals unambiguously identify B. anthracis versus other Bacillus species.
- Figure 9 is an ESI-TOF of a B. anthracis synthetic 16S_1228 duplex (reverse and forward strands). The technique easily distinguishes between the forward and reverse strands.
- Figure 10 is an ESI-FTICR-MS of a synthetic B. anthracis 16S_1337 46 base pair duplex.
- Figure 11 is an ESI-TOF -MS ofa 56mer oligonucleotide (3 scans) from the B. anthracis saspB gene with an internal mass standard. The internal mass standards are designated by asterisks.
- Figure 12 is an ESI-TOF-MS of an internal standard with 5 mM TBA-TFA buffer showing that charge stripping with tributylammonium trifluoroacetate reduces the most abundant charge state from [M-8H+]8- to [M-3H+J3-.
- Figure 13 is a portion of a secondary structure defining database according to one embodiment of the present invention, where two examples of selected sequences are displayed graphically thereunder.
- Figure 14 is a three dimensional graph demonstrating the grouping of sample molecular weight according to species.
- Figure 15 is a three dimensional graph demonstrating the grouping of sample molecular weights according to species of virus and mammal infected.
- Figure 16 is a three dimensional graph demonstrating the grouping of sample molecular weights according to species of virus, and animal-origin of infectious agent.
- Figure 17 is a figure depicting how the triangulation method of the present invention provides for the identification of an unknown bioagent without prior knowledge of the unknown agent.
- the use of different primer sets to distinguish and identify the unknown is also depicted as primer sets I, II and III within this figure.
- a three dimensional graph depicts all of bioagent space (170), including the unknown bioagent, which after use of primer set I (171) according to a method according to the present invention further differentiates and classifies bioagents according to major classifications (176) which, upon further analysis using primer set II (172) differentiates the unknown agent (177) from other, known agents (173) and finally, the use of a third primer set (175) further specifies subgroups within the family of the unknown (174).
- Figure 18 shows a representative mass spectra from amplicons derived from a single primer pair used in a dilution to extinction experiment with Bti spores as described. The concentration of spores is shown at the right of each spectrum. Positive and negative strands are labeled.
- Figure 19 shows a representative ROC curve demonstrating that air samples spiked with a spore sample are identified and detectable above background bioagents by using the present methods.
- the present invention provides, mter alia, methods for detection and identification of bioagents in an unbiased manner using "bioagent identifying amplicons.”
- "Intelligent primers” are selected to hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions to yield a bioagent identifying amplicon which can be amplified and which is amenable to molecular mass determination.
- the molecular mass then provides a means to uniquely identify the bioagent without a requirement for prior knowledge of the possible identity of the bioagent.
- the molecular mass or corresponding "base composition signature" (BCS) of the amplification product is then matched against a database of molecular masses or base composition signatures.
- the method can be applied to rapid parallel "multiplex" analyses, the results of which can be employed in a triangulation identification strategy.
- the present method provides rapid throughput and does not require nucleic acid sequencing of the amplified target sequence for bioagent detection and identification.
- a “bioagent” is any organism, cell, or virus, living or dead, or a nucleic acid derived from such an organism, cell or virus.
- bioagents include, but are not limited, to cells, including but not limited to, cells, including but not limited to human clinical samples, bacterial cells and other pathogens) viruses, fungi, and protists, parasites, and pathogenicity markers (including but not limited to: pathogenicity islands, antibiotic resistance genes, virulence factors, toxin genes and other bioregulating compounds).
- Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or spores) and may be encapsulated or bioengineered.
- a "pathogen” is a bioagent which causes a disease or disorder.
- Bacteria Despite enormous biological diversity, all forms of life on earth share sets of essential, common features in their genomes. Bacteria, for example have highly conserved sequences in a variety of locations on their genomes. Most notable is the universally conserved region of the ribosome. but there are also conserved elements in other non-coding RNAs, including RNAse P and the signal recognition particle (SRP) among others. Bacteria have a common set of absolutely required genes. About 250 genes are present in all bacterial species (Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 10268; Science, 1995, 270, 397), including tiny genomes like Mycoplasma, Ureaplasma and Rickettsia.
- genes encode proteins involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like.
- proteins are DNA polymerase III beta, elongation factor TU, heat shock protein groEL, RNA polymerase beta, phosphoglycerate kinase, NADH dehydrogenase, DNA ligase, DNA topoisomerase and elongation factor G.
- Operons can also be targeted using the present method.
- One example of an operon is the bfp operon from enteropathogenic E. coli.
- Multiple core chromosomal genes can be used to classify bacteria at a genus or genus species level to determine if an organism has threat potential.
- the methods can also be used to detect pathogenicity markers (plasmid or chromosomal) and antibiotic resistance genes to confirm the threat potential of an organism and to direct countermeasures.
- At least one polynucleotide segment is amplified to facilitate detection and analysis in the process of identifying the bioagent.
- nucleic acid segments which provide enough variability to distinguish each individual bioagent and whose molecular masses are amenable to molecular mass determination are herein described as "bioagent identifying amplicons.”
- amplicon refers to a segment of a polynucleotide which is amplified in an amplification reaction.
- intelligent primers are primers that are designed to bind to highly conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and yield amplification products which ideally provide enough variability to distinguish each individual bioagent, and which are amenable to molecular mass analysis.
- highly conserved it is meant that the sequence regions exhibit between about 80-100%, or between about 90-100%, or between about 95-100% identity.
- the molecular mass ofa given amplification product provides a means of identifying the bioagent from which it was obtained, due to the variability of the variable region.
- design of intelligent primers requires selection of a variable region with appropriate variability to resolve the identity ofa given bioagent.
- Bioagent identifying amplicons are ideally specific to the identity of the bioagent.
- a plurality of bioagent identifying amplicons selected in parallel for distinct bioagents which contain the same conserved sequences for hybridization of the same pair of intelligent primers are herein defined as "correlative bioagent identifying amplicons.”
- the bioagent identifying amplicon is a portion of a ribosomal RNA (rRNA) gene sequence.
- Ribosomal RNA (rRNA) genes comprise regions that provide useful base composition signatures. Like many genes involved in core life functions, rRNA genes contain sequences that are extraordinarily conserved across bacterial domains interspersed with regions of high variability that are more specific to each species. The variable regions can be utilized to build a database of base composition signatures. The strategy involves creating a structure-based alignment of sequences of the small (16S) and the large (23 S) subunits of the rRNA genes.
- regions that are useful as bioagent identifying amplicons include: a) between about 80 and 100%, or greater than about 95% identity among species of the particular bioagent of interest, of upstream and downstream nucleotide sequences which serve as sequence amplification primer sites; b) an intervening variable region which exhibits no greater than about 5% identity among species; and c) a separation of between about 30 and 1000 nucleotides, or no more than about 50-250 nucleotides, or no more than about 60-100 nucleotides, between the conserved regions.
- the conserved sequence regions of the chosen bioagent identifying amplicon must be highly conserved among all Bacillus species while the variable region of the bioagent identifying amplicon is sufficiently variable such that the molecular masses of the amplification products of all species of Bacillus are distinguishable.
- Bioagent identifying amplicons amenable to molecular mass determination are either of a length, size or mass compatible with the particular mode of molecular mass determination or compatible with a means of providing a predictable fragmentation pattern in order to obtain predictable fragments of a length compatible with the particular mode of molecular mass determination.
- Such means of providing a predictable fragmentation pattern of an amplification product include, but are not limited to, cleavage with restriction enzymes or cleavage primers, for example.
- bioagent division is defined as group of bioagents above the species level and includes but is not limited to: orders, families, classes, clades, genera or other such groupings of bioagents above the species level.
- members of the Bacillus/Clostridia group or gamma-proteobacteria group may be identified as such by employing broad range survey intelligent primers such as primers which target 16S or 23S ribosomal RNA.
- broad range survey intelligent primers are capable of identification of bioagents at the species level.
- One main advantage of the detection methods of the present invention is that the broad range survey intelligent primers need not be specific for a particular bacterial species, or even genus, such as Bacillus or Streptomyces. Instead, the primers recognize highly conserved regions across hundreds of bacterial species including, but not limited to, the species described herein. Thus, the same broad range survey intelligent primer pair can be used to identify any desired bacterium because it will bind to the conserved regions that flank a variable region specific to a single species, or common to several bacterial species, allowing unbiased nucleic acid amplification of the intervening sequence and determination of its molecular weight and base composition.
- primers used in the present method bind to one or more of these regions or portions thereof.
- flanking rRNA primer sequences serve as good intelligent primer binding sites to amplify the nucleic acid region of interest for most, if not all, bacterial species.
- the intervening region between the sets of primers varies in length and/or composition, and thus provides a unique base composition signature. Examples of intelligent primers that amplify regions of the 16S and 23 S rRNA are shown in Figures 1A-1H.
- a typical primer amplified region in 16S rRNA is shown in Figure 2.
- the arrows represent primers that bind to highly conserved regions which flank a variable region in 16S rRNA domain III.
- the amplified region is the stem-loop structure under "1100-1188." It is advantageous to design the broad range survey intelligent primers to minimize the number of primers required for the analysis, and to allow detection of multiple members of a bioagent division using a single pair of primers.
- the advantage of using broad range survey intelligent primers is that once a bioagent is broadly identified, the process of further identification at species and sub-species levels is facilitated by directing the choice of additional intelligent primers.
- 5 "Division- wide" intelligent primers are designed with an objective of identifying a bioagent at the species level. As a non-limiting example, a Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis can be distinguished from each other using division- wide intelligent primers. Division-wide intelligent primers are not always required for identification at the species level because broad range survey intelligent primers may provide sufficient identification
- Drill-down intelligent primers are designed with an objective of identifying a subspecies characteristic of a bioagent.
- a "sub-species characteristic” is defined as a property imparted to a bioagent at the sub-species level of identification as a result of the presence or absence of a particular segment of nucleic acid. Such sub-species characteristics include, but are
- 25 oligonucleotide primers can be designed such that the nucleotide corresponding to this position is a base which can bind to more than one nucleotide, referred to herein as a "universal base.”
- a base which can bind to more than one nucleotide
- inosine (I) binds to U, C or A
- guanine (G) binds to U or C
- uridine (U) binds to U or C.
- Other examples of universal bases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al, Nucleosides and Nucleotides, 1995, 14, 1001-
- nucleotides dP or dK Hill et al
- an acyclic nucleoside analog containing 5-nitroindazole Van Aerschot et al, Nucleosides and Nucleotides, 1995, 14, 1053-1056
- purine analog l-(2-deoxy- ⁇ -D-ribofuranosyl)-imidazole-4-carboxamide Sala et al, Nucl. Acids Res., 1996, 24, 3302-3306).
- the oligonucleotide primers are designed such that the first and second positions of each triplet are occupied by nucleotide analogs which bind with greater affinity than the unmodified nucleotide.
- these analogs include, but are not limited to, 2,6-diaminopurine which binds to thymine, propyne T which binds to adenine and propyne C and phenoxazines, including G-clamp, which binds to G.
- Propynylated pyrimidines are described in U.S. Patent Nos.
- a theoretically ideal bioagent detector would identify, quantify, and report the complete nucleic acid sequence of every bioagent that reached the sensor.
- the complete sequence of the nucleic acid component of a pathogen would provide all relevant information about the threat, including its identity and the presence of drug-resistance or pathogenicity markers. This ideal has not yet been achieved.
- the present invention provides a straightforward strategy for obtaining information with the same practical value based on analysis of bioagent identifying amplicons by molecular mass determination. In some cases, a molecular mass ofa given bioagent identifying amplicon alone does not provide enough resolution to unambiguously identify a given bioagent.
- the molecular mass of the bioagent identifying amplicon obtained using the intelligent primer pair "16S 971" would be 55622 Da for both E. coli and Salmonella typhimurium.
- additional intelligent primers are employed to analyze additional bioagent identifying amplicons, a "triangulation identification” process is enabled.
- the "16S_1100” intelligent primer pair yields molecular masses of 55009 and 55005 Da for E. coli and Salmonella typhimurium, respectively.
- the "23S_855" intelligent primer pair yields molecular masses of 42656 and 42698 Da for E. coli and Salmonella typhimurium, respectively.
- the second and third intelligent primer pairs provided the additional "fingerprinting" capability or resolution to distinguish between the two bioagents.
- the triangulation identification process is pursued by measuring signals from a plurality of bioagent identifying amplicons selected within multiple core genes. This process is used to reduce false negative and false positive signals, and enable reconstruction of the origin of hybrid or otherwise engineered bioagents.
- this process after identification of multiple core genes, alignments are created from nucleic acid sequence databases. The alignments are then analyzed for regions of conservation and variation, and bioagent identifying amplicons are selected to distinguish bioagents based on specific genomic differences. For example, identification of the three part toxin genes typical of J?. anthracis (Bowen et al, J. Appl. Microbiol, 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.
- the triangulation identification process can be pursued by characterization of bioagent identifying amplicons in a massively parallel fashion using the polymerase chain reaction (PCR), such as multiplex PCR, and mass spectrometric (MS) methods. Sufficient quantities of nucleic acids should be present for detection of bioagents by MS.
- PCR requires one or more pairs of oligonucleotide primers that bind to regions which flank the target sequence(s) to be amplified. These primers prime synthesis ofa different strand of DNA, with synthesis occurring in the direction of one primer towards the other primer.
- the primers, DNA to be amplified, a thermostable DNA polymerase (e.g. Taq polymerase), the four deoxynucleotide triphosphates, and a buffer are combined to initiate DNA synthesis.
- the solution is denatured by heating, then cooled to allow annealing of newly added primer, followed by another round of DNA synthesis. This process is typically repeated for about 30 cycles, resulting in amplification of the target sequence.
- PCR ligase chain reaction
- SDA strand displacement amplification
- the high-resolution MS technique allows separation of bioagent spectral lines from background spectral lines in highly cluttered environments.
- the detection scheme for the PCR products generated from the bioagent(s) incorporates at least three features. First, the technique simultaneously detects and differentiates multiple (generally about 6-10) PCR products. Second, the technique provides a molecular mass that uniquely identifies the bioagent from the possible primer sites. Finally, the detection technique is rapid, allowing multiple PCR reactions to be run in parallel.
- Mass spectrometry (MS)-based detection of PCR products provides a means for determination of BCS which has several advantages.
- MS is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product is identified by its molecular mass.
- the current state of the art in mass spectrometry is such that less than femtomole quantities of material can be readily analyzed to afford information about the molecular contents of the sample.
- An accurate assessment of the molecular mass of the material can be quickly obtained, irrespective of whether the molecular weight of the sample is several hundred, or in excess of one hundred thousand atomic mass units (amu) or Daltons.
- Intact molecular ions can be generated from amplification products using one of a variety of ionization techniques to convert the sample to gas phase. These ionization methods include, but are not limited to, electrospray ionization (ES), matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB).
- ES electrospray ionization
- MALDI matrix-assisted laser desorption ionization
- FAB fast atom bombardment
- MALDI of nucleic acids along with examples of matrices for use in MALDI of nucleic acids, are described in WO 98/54751 (Genetrace, Inc.).
- large DNAs and RNAs, or large amplification products therefrom can be digested with restriction endonucleases prior to ionization.
- restriction endonucleases for example, an amplification product that was 10 kDa could be digested with a series of restriction endonucleases to produce a panel of, for example, 100 Da fragments.
- Restriction endonucleases and their sites of action are well known to the skilled artisan.
- mass spectrometry can be performed for the purposes of restriction mapping. Upon ionization, several peaks are observed from one sample due to the formation of ions with different charges. Averaging the multiple readings of molecular mass obtained from a single mass spectrum affords an estimate of molecular mass of the bioagent.
- Electrospray ionization mass spectrometry is particularly useful for very high molecular weight polymers such as proteins and nucleic acids having molecular weights greater than 10 kDa, since it yields a distribution of multiply-charged molecules of the sample without causing a significant amount of fragmentation.
- the mass detectors used in the methods of the present invention include, but are not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, and triple quadrupole.
- FT-ICR-MS Fourier transform ion cyclotron resonance mass spectrometry
- ion trap ion trap
- TOF time of flight
- Q-TOF Q-TOF
- triple quadrupole triple quadrupole
- the mass spectrometric techniques which can be used in the present invention include, but are not limited to, tandem mass spectrometry, infrared multiphoton dissociation and pyrolytic gas chromatography mass spectrometry (PGC-MS).
- the bioagent detection system operates continually in bioagent detection mode using pyrolytic GC-MS without PCR for rapid detection of increases in biomass (for example, increases in fecal contamination of drinking water or of germ warfare agents).
- biomass for example, increases in fecal contamination of drinking water or of germ warfare agents.
- a continuous sample stream flows directly into the PGC-MS combustion chamber.
- a PCR process is automatically initiated.
- Bioagent presence produces elevated levels of large molecular fragments from, for example, about 100-7,000 Da which are observed in the PGC-MS spectrum.
- the observed mass spectrum is compared to a threshold level and when levels of biomass are determined to exceed a predetermined threshold, the bioagent classification process described hereinabove (combining PCR and MS, such as FT-ICR MS) is initiated.
- alarms or other processes are also initiated by this detected biomass level.
- the accurate measurement of molecular mass for large DNAs is limited by the adduction of cations from the PCR reaction to each strand, resolution of the isotopic peaks from natural abundance 13 C and 15 N isotopes, and assignment of the charge state for any ion.
- the cations are removed by in-line dialysis using a flow-through chip that brings the solution containing the PCR products into contact with a solution containing ammonium acetate in the presence of an electric field gradient orthogonal to the flow.
- the latter two problems are addressed by operating with a resolving power of > 100,000 and by incorporating isotopically depleted nucleotide triphosphates into the DNA.
- the resolving power of the instrument is also a consideration. At a resolving power of 10,000, the modeled signal from the [M-14H+] 14" charge state of an 84mer PCR product is poorly characterized and assigmnent of the charge state or exact mass is impossible. At a resolving power of 33,000, the peaks from the individual isotopic components are visible. At a resolving power of 100,000, the isotopic peaks are resolved to the baseline and assignment of the charge state for the ion is straightforward.
- the [ 13 C, 15 N] -depleted triphosphates are obtained, for example, by growing microorganisms on depleted media and harvesting the nucleotides (Batey et al, Nucl. Acids Res., 1992, 20, 4515-4523). While mass measurements of intact nucleic acid regions are believed to be adequate to determine most bioagents, tandem mass spectrometry (MS n ) techniques may provide more definitive information pertaining to molecular identity or sequence. Tandem MS involves the coupled use of two or more stages of mass analysis where both the separation and detection steps are based on mass spectrometry. The first stage is used to select an ion or component of a sample from which further structural information is to be obtained.
- the selected ion is then fragmented using, e.g., blackbody irradiation, infrared multiphoton dissociation, or collisional activation.
- ions generated by electrospray ionization can be fragmented using IR multiphoton dissociation. This activation leads to dissociation of glycosidic bonds and the phosphate backbone, producing two series of fragment ions, called the w-series (having an intact 3' terminus and a 5' phosphate following internal cleavage) and the ⁇ -Base series(having an intact 5' terminus and a 3' furan).
- the second stage of mass analysis is then used to detect and measure the mass of these resulting fragments of product ions.
- Such ion selection followed by fragmentation routines can be performed multiple times so as to essentially completely dissect the molecular sequence ofa sample.
- a nucleotide analog or "tag” is incorporated during amplification (e.g., a 5- (trifluoromethyl) deoxythymidine triphosphate) which has a different molecular weight than the unmodified base so as to improve distinction of masses.
- tags are described in, for example, PCT WO97/33000, which is incorporated herein by reference in its entirety. This further limits the number of possible base compositions consistent with any mass. For example, 5-
- (trifluoromethyl)deoxythymidine triphosphate can be used in place of dTTP in a separate nucleic acid amplification reaction. Measurement of the mass shift between a conventional amplification product and the tagged product is used to quantitate the number of thymidine nucleotides in each of the single strands. Because the strands are complementary, the number of adenosine nucleotides in each strand is also determined.
- the number of G and C residues in each strand is determined using, for example, the cytidine analog 5-methylcytosine (5-meC) or propyne C.
- the mass tag phosphorothioate A (A*) was used to distinguish a Bacillus anthracis cluster.
- the B. anthracis (A 1 G 9 C 1 T 9 ) had an average MW of 14072.26, and the B. anthracis (A 1 A* 13 G C ⁇ T ) had an average molecular weight of 14281.11 and the phosphorothioate A had an average molecular weight of +16.06 as determined by ESI-TOF MS.
- the deconvoluted spectra are shown in Figure 5.
- the measured molecular masses of each strand are 30,000.115Da and 31,000.115 Da respectively, and the measured number of dT and dA residues are (30,28) and (28,30). If the molecular mass is accurate to 100 ppm, there are 7 possible combinations of dG+dC possible for each strand. However, if the measured molecular mass is accurate to 10 ppm, there are only 2 combinations of dG+dC, and at 1 ppm accuracy there is only one possible base composition for each strand.
- Signals from the mass spectrometer may be input to a maximum-likelihood detection and classification algorithm such as is widely used in radar signal processing.
- the detection processing uses matched filtering of BCS observed in mass-basecount space and allows for detection and subtraction of signatures from known, harmless organisms, and for detection of unknown bioagent threats. Comparison of newly observed bioagents to known bioagents is also possible, for estimation of threat level, by comparing their BCS to those of known organisms and to known forms of pathogenicity enhancement, such as insertion of antibiotic resistance genes or toxin genes. Processing may end with a Bayesian classifier using log likelihood ratios developed from the observed signals and average background levels.
- the program emphasizes performance predictions culminating in probability-of-detection versus probability-of-false-alarm plots for conditions involving complex backgrounds of naturally occurring organisms and environmental contaminants.
- Matched filters consist of a priori expectations of signal values given the set of primers used for each of the bioagents.
- a genomic sequence database e.g. GenBank
- GenBank is used to define the mass basecount matched filters.
- the database contains known threat agents and benign background organisms. The latter is used to estimate and subtract the signature produced by the background organisms.
- a maximum likelihood detection of known background organisms is implemented using matched filters and a running-sum estimate of the noise covariance.
- a base composition signature is the exact base composition determined from the molecular mass of a bioagent identifying amplicon.
- a BCS provides an index ofa specific gene in a specific organism.
- Base compositions like sequences, vary slightly from isolate to isolate within species. It is possible to manage this diversity by building "base composition probability clouds” around the composition constraints for each species. This permits identification of organisms in a fashion similar to sequence analysis. A "pseudo four-dimensional plot" can be used to visualize the concept of base composition probability clouds ( Figure 18).
- Optimal primer design requires optimal choice of bioagent identifying amplicons and maximizes the separation between the base composition signatures of individual bioagents. Areas where clouds overlap indicate regions that may result in a misclassification, a problem which is overcome by selecting primers that provide information from different bioagent identifying amplicons, ideally maximizing the separation of base compositions.
- one aspect of the utility of an analysis of base composition probability clouds is that it provides a means for screening primer sets in order to avoid potential misclassifications of BCS and bioagent identity.
- Another aspect of the utility of base composition probability clouds is that they provide a means for predicting the identity ofa bioagent whose exact measured BCS was not previously observed and/or indexed in a BCS database due to evolutionary transitions in its nucleic acid sequence.
- the present invention provides bioagent classifying information similar to DNA sequencing and phylogenetic analysis at a level sufficient to detect and identify a given bioagent. Furthermore, the process of determination ofa previously unknown BCS for a given bioagent (for example, in a case where sequence information is unavailable) has downstream utility by providing additional bioagent indexing information with which to populate BCS databases. The process of future bioagent identification is thus greatly improved as more BCS indexes become available in the BCS databases.
- Another embodiment of the present invention is a method of surveying bioagent samples that enables detection and identification of all bacteria for which sequence information is available using a set of twelve broad-range intelligent PCR primers.
- Six of the twelve primers are "broad range survey primers" herein defined as primers targeted to broad divisions of bacteria (for example, the Bacillus/Clostridia group or gamma-proteobacteria).
- the other six primers of the group of twelve primers are "division- wide” primers herein defined as primers which provide more focused coverage and higher resolution. This method enables identification of nearly 100% of known bacteria at the species level.
- a further example of this embodiment of the present invention is a method herein designated "survey/drill-down" wherein a subspecies characteristic for detected bioagents is obtained using additional primers.
- a subspecies characteristic include but are not limited to: antibiotic resistance, pathogenicity island, virulence factor, strain type, sub-species type, and clade group.
- bioagent detection, confirmation and a subspecies characteristic can be provided within hours.
- the survey/drill-down method can be focused to identify bioengineering events such as the insertion of a toxin gene into a bacterial species that does not normally make the toxin.
- the present methods allow extremely rapid and accurate detection and identification of bioagents compared to existing methods. Furthermore, this rapid detection and identification is possible even when sample material is impure.
- the methods leverage ongoing biomedical research in virulence, pathogenicity, drug resistance and genome sequencing into a method which provides greatly improved sensitivity, specificity and reliability compared to existing methods, with lower rates of false positives.
- the methods are useful in a wide variety of fields, including, but not limited to, those fields discussed below.
- the methods disclosed herein can be used for environmental testing.
- "Environment” is herein defined as including both natural environment such as soil, water, living matter such as plants, as well as environments created by humans such as buildings, vehicles, containers, water towers. Detection and discrimination of pathogenic vs. non-pathogenic bacteria, viruses, parasites, fungi and the like, in samples of water, land, air, or other samples, can be carried out.
- Water samples can be obtained from, for example, lakes, rivers, oceans, streams, water treatment systems, rainwater, groundwater, water table, reservoirs, wells, bottled water, and the like.
- Air samples can be obtained from ventilation systems, airplane cabins, schools, hospitals, mass transit locations such as subways, train stations, airports, and the like. Land samples can be obtained from any location.
- the methods disclosed herein can be used for detecting the presence of bioagents in a container, such as a package, box, envelope, mail tube, railroad box car, and the like.
- a container such as a package, box, envelope, mail tube, railroad box car, and the like.
- mail and package delivery entities and agencies both domestic and abroad, as well investigative agencies such as the FBI and ATF can use the present methods to detect bioagents in containers. Appropriate sampling techniques are well known to those skilled in the art.
- the methods disclosed herein can be used for detecting the presence of pathogenic and non-pathogenic bacteria, viruses, parasites, fungi and the like in products.
- "Products” are defined as objects for consumption such as processed food, drinks and cosmetics.
- food and wine can be examined for the presence of pathogenic and non-pathogenic bacteria, viruses, parasites, fungi and the like.
- Particular types of foods susceptible to bioagent contamination such as agricultural products, meat products and eggs, can be examined for pathogenic organisms such as E. coli and Salmonella species.
- Such examination procedures can be used by, for example, the wholesalers of foodstuffs and beverages, or by regulatory agencies such as the U.S. Department of Agriculture and the Food and Drug Administration.
- grapes and wines can be examined using the present methods to detect particular strains of bacteria or yeast that may indicate a particular time upon which to harvest the grapes or alter the wine-making process.
- Appropriate methods of sampling food, drink and cosmetic products are well known to those will skill in the art.
- the present invention can be used for rapid detection and identification of species of household mold which is becoming a growing concern for homeowners. Methods of sampling household molds are well known to those skilled in the art.
- the methods of the present invention can be used to monitor bioremediation processes by detection, identification and quantification of indigenous and bioremediating bioagents.
- Methods of sampling sites of bioremediation include, but are not limited to, water and soil sampling methods which are well known to those skilled in the art. While the present invention has been described with specificity in accordance with certain of its embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.
- nucleic acid is isolated from the organisms and amplified by PCR using standard methods prior to BCS determination by mass spectrometry.
- Nucleic acid is isolated, for example, by detergent lysis of bacterial cells, centrifugation and ethanol precipitation. Nucleic acid isolation methods are described in, for example, Current Protocols in Molecular Biology (Ausubel et al.) and Molecular Cloning; A Laboratory Manual (Sambrook et al).
- the nucleic acid is then amplified using standard methodology, such as PCR, with primers which bind to conserved regions of the nucleic acid which contain an intervening variable sequence as described below.
- Swab Sample Protocol Allegiance S/P brand culture swabs and collection/transport system are used to collect samples. After drying, swabs are placed in 17x100 mm culture tubes (VWR International) and the genomic nucleic acid isolation is carried out automatically with a Qiagen Mdx robot and the Qiagen QIAamp DNA Blood BioRobot Mdx genomic preparation kit (Qiagen, Valencia, CA).
- the FTICR instrument is based on a 7 tesla actively shielded superconducting magnet and modified Bruker Daltonics Apex II 70e ion optics and vacuum chamber.
- the spectrometer is interfaced to a LEAP PAL autosampler and a custom fluidics control system for high throughput screening applications. Samples are analyzed directly from 96-well or 384-well microtiter plates at a rate of about 1 sample/minute.
- the Bruker data- acquisition platform is supplemented with a lab-built ancillary NT datastation which controls the autosampler and contains an arbitrary waveform generator capable of generating complex rf- excite waveforms (frequency sweeps, filtered noise, stored waveform inverse Fourier transform (SWIFT), etc.) for sophisticated tandem MS experiments.
- a lab-built ancillary NT datastation which controls the autosampler and contains an arbitrary waveform generator capable of generating complex rf- excite waveforms (frequency sweeps, filtered noise, stored waveform inverse Fourier transform (SWIFT), etc.) for sophisticated tandem MS experiments.
- typical performance characteristics include mass resolving power in excess of 100,000 (FWHM), low ppm mass measurement errors, and an operable m/z range between 50 and 5000 m/z.
- Modified ESI Source In sample-limited analyses, analyte solutions are delivered at 150 nL/minute to a 30 mm i.d. fused-silica ESI emitter mounted on a 3-D micromanipulator.
- the ESI ion optics consists of a heated metal capillary, an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode.
- the 6.2 cm rf-only hexapole is comprised of 1 mm diameter rods and is operated at a voltage of 380 Vpp at a frequency of 5 MHz.
- a lab-built electro-mechanical shutter can be employed to prevent the electrospray plume from entering the inlet capillary unless triggered to the "open" position via a TTL pulse from the data station.
- a stable electrospray plume is maintained between the ESI emitter and the face of the shutter.
- the back face of the shutter arm contains an elastomeric seal that can be positioned to form a vacuum seal with the inlet capillary. When the seal is removed, a 1 mm gap between the shutter blade and the capillary inlet allows constant pressure in the external ion reservoir regardless of whether the shutter is in the open or closed position.
- a "time slice" of ions is allowed to enter the inlet capillary and is subsequently accumulated in the external ion reservoir.
- the rapid response time of the ion shutter ( ⁇ 25 ms) provides reproducible, user defined intervals during which ions can be injected into and accumulated in the external ion reservoir.
- a 25 watt CW CO 2 laser operating at 10.6 ⁇ m has been interfaced to the spectrometer to enable infrared multiphoton dissociation (IRMPD) for oligonucleotide sequencing and other tandem MS applications.
- IRMPD infrared multiphoton dissociation
- An aluminum optical bench is positioned approximately 1.5 m from the actively shielded superconducting magnet such that the laser beam is aligned with the central axis of the magnet.
- the unfocused 3 mm laser beam is aligned to traverse directly through the 3.5 mm holes in the trapping electrodes of the FTICR trapped ion cell and longitudinally traverse the hexapole region of the external ion guide finally impinging on the skimmer cone.
- This scheme allows IRMPD to be conducted in an m/z selective manner in the trapped ion cell (e.g. following a SWIFT isolation of the species of interest), or in a broadband mode in the high pressure region of the external ion reservoir where collisions with neutral molecules stabilize IRMPD-generated metastable fragment ions resulting in increased fragment ion yield and sequence coverage.
- Table 2 shows a small cross section of a database of calculated molecular masses for over 9 primer sets and approximately 30 organisms.
- the primer sets were derived from rRNA alignment. Examples of regions from rRNA consensus alignments are shown in Figures lA-lC. Lines with arrows are examples of regions to which intelligent primer pairs for PCR are designed.
- the primer pairs are >95% conserved in the bacterial sequence database (currently over 10,000 organisms).
- the intervening regions are variable in length and/or composition, thus providing the base composition "signature" (BCS) for each organism.
- Primer pairs were chosen so the total length of the amplified region is less than about 80-90 nucleotides.
- the label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram.
- the two strands differ by two (AT-»CG) substitutions, and have measured masses of 14206.396 and 14208.373 + 0.010 Da, respectively.
- the possible base compositions derived from the masses of the forward and reverse strands for the B. anthracis products are listed in Table 3.
- compositions for the forward strand and the 18 compositions for the reverse strand that were calculated, only one pair (shown in bold) are complementary, corresponding to the actual base compositions of the B. anthracis PCR products.
- Example 4 BCS of Region from Bacillus anthracis and Bacillus cereus
- the pathogen Vibrio cholera can be distinguished from Vibrio parahemolyticus with ⁇ M > 600 Da using one of three 16S primer sets shown in Table 2 (16S_971, 16S_1228 or 16S_1294) as shown in Table 4.
- the two mycoplasma species in the list (M. genitalium and M. pneumoniae) can also be distinguished from each other, as can the three mycobacteriae. While the direct mass measurements of amplified products can identify and distinguish a large number of organisms, measurement of the base composition signature provides dramatically enhanced resolving power for closely related organisms.
- compositional analysis or fragmentation patterns are used to resolve the differences.
- the single base difference between the two organisms yields different fragmentation patterns, and despite the presence of the ambiguous/unidentified base N at position 20 in B. anthracis, the two organisms can be identified.
- Tables 4a-b show examples of primer pairs from Table 1 which distinguish pathogens from background. Table 4a
- Table 5 shows the expected molecular weight and base composition of region
- Table 6 shows base composition (single strand) results for 16S_1100-1188 primer amplification reactions different species of bacteria. Species which are repeated in the table (e.g., Clostridium botulinum) are different strains which have different base compositions in the 16S_1100-1188 region.
- the same organism having different base compositions are different strains. Groups of organisms which are highlighted or in italics have the same base compositions in the amplified region. Some of these organisms can be distinguished using multiple primers. For example, Bacillus anthracis can be distinguished from Bacillus cereus and Bacillus thuringiensis using the primer 16S_971-1062 (Table 7). Other primer pairs which produce unique base composition signatures are shown in Table 6 (bold). Clusters containing very similar threat and ubiquitous non-threat organisms (e.g. anthracis cluster) are distinguished at high resolution with focused sets of primer pairs. The known biowarfare agents in Table 6 are Bacillus anthracis, Yersinia pestis, Francisella tularensis and Rickettsia prowazekii. Table 7
- B. anthracis has an ambiguous base at position 20.
- the mass measurement accuracy that can be obtained using an internal mass standard in the ESI-MS study of PCR products is shown in Fig.8.
- the mass standard was a 20-mer phosphorothioate oligonucleotide added to a solution containing a 56-mer PCR product from the B. anthracis spore coat protein sspE.
- the mass of the expected PCR product distinguishes B. anthracis from other species of Bacillus such as B. thuringiensis and B. cereus.
- ESI-TOF MS spectra were obtained on a synthetic 56-mer oligonucleotide (5 ⁇ M) from the saspB gene of B. anthracis containing an internal mass standard at an ESI of 1.7 ⁇ L/min as a function of sample consumption.
- the results show that the signal to noise is improved as more scans are summed, and that the standard and the product are visible after only 100 scans.
- Example 10 ESI-TOF MS of an Internal Standard with Tributylammonium (TBA)- trifluoroacetate (TFA) Buffer An ESI-TOF-MS spectrum of a 20-mer phosphorothioate mass standard was obtained following addition of 5 mM TBA-TFA buffer to the solution. This buffer strips charge from the oligonucleotide and shifts the most abundant charge state from [M-8H + ] 8" to [M-3H + ] 3" (Fig. 12).
- the molecular masses obtained through Examples 1-10 are compared to molecular masses of known bioagents stored in a master database to obtain a high probability matching molecular mass.
- Example 11 The same procedure as in Example 11 is followed except that the local computer did not store the Master database.
- the Master database is interrogated over an internet connection, searching for a molecular mass match.
- the local computer is connected to the internet and has the ability to store a master database locally.
- the local computer system periodically, or at the user's discretion, interrogates the Master database, synchronizing the local master database with the global Master database. This provides the current molecular mass information to both the local database as well as to the global Master database. This further provides more of a globalized knowledge base.
- Example 15 Environmental Sampling Protocol for Anthrax Spores Using Non-Cotton Swabs
- the use of non-cotton, sterile swabs to collect environmental samples is the preferred method because it reduces the amount of normal background contamination and improves the recovery of anthrax spores (see, for example, www.dhmh.state.md.us/labs/html/Terrorism/Biological/BT_env_samp_prtcl.html).
- the following protocol is used to collect samples from small non-porous surfaces or objects for culture and identification of Bacillus anthracis.
- Non-powdered gloves, a disposable gown, and facemask are worn during the collection of swab samples from the environment.
- a sterile Dacron or Rayon dry swab (non-cotton swab) is used to collect environmental samples.
- a swab moistened with sterile saline or distilled water is used to collect samples from computer keyboards, desks, mail sorting areas, and other sites within a building or work facility.
- a separate swab is used for each site.
- a swab is taken of a 10 x 10 inch square area per swab.
- Each swab is placed in a 15 ml sterile tube, the shaft of swab is snapped off at the lip of the tube, and the tube is closed.
- Each tube is labeled appropriately and placed in a self-sealable plastic bag. Multiple tubes can be placed in the same bag. Each tube is labeled with the date, facility location, and sampling site. The outside of the sealed bag is cleaned by wiping with 10% bleach solution. Place clean, sealed bags into an unused similar self-sealing bag before delivery. Appropriate chain-of-custody documentation and procedure is constantly maintained.
- Soil cores are collected to a depth of 8 cm, using an 8 cm diameter soil corer. Soils for analysis of root biomass and composition are collected separately. A single 3.5 cm diameter core is collected to a depth of 16 cm. Three 3.5 cm cores are also taken. These cores are divided into 0-1, 1-8, and 8-16 cm depth increments. Polyethylene bags containing soil cores are handled to minimize disturbance and changes in soil moisture and temperature. While sampling, any unusual features of the area where the core was taken are noted. Microbial Group soil cores are sieved (4 mm) and the plant material discarded.
- Example 17 Estimation of the Sensitivity of Detection of Spores in Water Samples
- Example 18 Identification of Bioagents in air samples.
- Multi-hour air samples from indoor or outdoor air were obtained using a Spin-Con sampler which can sample up to 18 x 10 6 L of air.
- the air samples were processed as 10 ml samples and spiked with Bacillis anthracis spores or Bacillis thuringiensis israeliensis (Bti) spores.
- the samples were filtered and lysed via bead-beating.
- Nucleic acids were isolated from the samples by standard methods and amplified by PCR using intelligent primers. Samples were then desalted and analyzed by FT-ICR mass spectrometry according to methods outlined in example 2.
- Figure 19 is an ROC curve that indicates that a spore spiked sample (Pd sample threat) is easily distinguished from background bioagents (Pfa sample threat and Pfa system). The data set indicates that the present method of air sample detection and identification of bioagents provides a reliable means of air sample surveillance.
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US20040180328A1 (en) | 2004-09-16 |
AU2003297997A1 (en) | 2004-06-30 |
US20040253583A1 (en) | 2004-12-16 |
AU2003297997A8 (en) | 2004-06-30 |
US20040121314A1 (en) | 2004-06-24 |
US7255992B2 (en) | 2007-08-14 |
WO2004053076A3 (en) | 2004-09-02 |
US20040253619A1 (en) | 2004-12-16 |
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