BioWatch PCR Assays Building Confidence, Ensuring Reliability: Abbreviated Version (2015) / Chapter Skim
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5 Looking to the Future: Multiplex PCR and Next Generation Sequencing
5 Looking to the Future: Multiplex PCR and Next Generation Sequencing
Pages 123-136
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From page 123... ...
; • Multiplex PCR using conserved primers in a single reaction to amplify a large number of related species for subsequent analysis (e.g., using primers directed toward 16S ribosomal DNA, which is capable of amplifying a wide range of bacteria, followed by sequencing of the amplification products to identify the organisms) ; • PCR amplification using random, universal or whole genome amplification followed by another, high-throughput method of analysis and readout, such as via microarrays; and • PCR amplicon, targeted probe enrichment, or metagenomic sequencing of various types.
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From page 124... ...
A Probe A Fluorescent Reporter Quencher Primer set 1 Fluorescent Probe B Reporter Quencher Primer set 2 Primer set 1 Fluorescent Probe C Reporter Quencher Primer set 3 Primer set 2 Primer set 3 B BOX FIGURE: Depiction of different strategies for multiplex PCR.
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From page 125... ...
The secondary assay for any of the tested BioWatch agents, which looks for the presence of three or more signatures, also is run in a singleplex manner, meaning that the "secondary assay" consists of three or more individual singleplex assays. However, if BioWatch assays were combined in a multiplex fashion, multiple targets could be tested at one time, potentially saving time and labor but with a potential loss of detection sensitivity.
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From page 126... ...
• Rather than designing one multiplex to cover all desired targets, it is also possible to design several multiplex assays that each covers a set of targets whose performance under PCR conditions can be better optimized; such multiplex assays can be used in combination. • Detection of a multiplex PCR typically requires more sophisti cated instrumentation than a singleplex, because multiple colors need to be detected when using fluorescent probes (see Box 5-1)
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From page 127... ...
However, several additional considerations arise in the development and validation of multiplex PCR assays. Just as in singleplex assays but on a larger scale, the inclusion of multiple pairs of primers and multiple probes into a single reaction mix can result in primer–primer interactions that interfere with target amplification or lead to the production of nonspecific amplification products and reduce assay sensitivity.
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From page 128... ...
Similarly, one or more representative targets of the multiplex assay can be used to demonstrate that carryover and cross- ontamination do not c occur in the device between samples, rather than testing all targets. Finally, the current FDA guidance indicates that a change to an approved multiplexed assay, such as the addition of a new target, requires the manufacturer to submit a 510(k)
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From page 129... ...
These approaches in concert with the FDA guidance documents should provide a suitable basis for validation testing of multiplexed PCR assays for BioWatch. The BioWatch program utilized actionable multiplex assays from late 2007 before withdrawing them from use in mid-2009.
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From page 130... ...
TAKING ACCOUNT OF STEP CHANGES IN TECHNOLOGY The BioWatch program currently operates under several assumptions and limitations. These include the use of a two-stage screening and secondary assay process based on real-time PCR of a nominal number of signature sequences, which places the emphasis on making these PCR assays as reliable and robust (as sensitive and as specific)
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From page 131... ...
Metagenomic sequencing could also be used now as part of efforts to better understand background genomic diversity on BioWatch filters from various collectors in various jurisdictions and at various time points. As discussed in Chapter 3, certain microbial strains may produce positive assay detections as a result of time, weather, or location-specific environmental sources and improving the ability to predict these circumstances would aid users in interpreting results.
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From page 132... ...
Sequencing as Confirmation of a PCR Screening Positive The primary issue is whether it is feasible to utilize metagenomic ("random" shotgun) NGS on a portion of a BioWatch filter to provide confirmation of a PCR screening positive result and to provide additional characterization of the material present in the sample.
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From page 133... ...
A Note About Targeted Versus Metagenomic NGS The above discussion assumed that metagenomic sequencing was performed on nucleic acids extracted from BioWatch filters. Commercial technologies exist that permit targeted sequencing, which can be accomplished via targeted-PCR methods (e.g., pools of PCR primers that amplify genomic regions that are highly discriminating)
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From page 134... ...
and Listeria spp. are being sequenced each month.1 These whole-genome sequencing approaches using NGS can also be applied by BioWatch in the future to recover additional genomes of biothreat agents and thereby establish more complete reference databases.
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From page 135... ...
TABLE 5-1 Applications of Next-Generation Sequencing for BioWatch Key Challenges / Barriers to Implementation Related Terms Purpose Advantages Complexity of Turnaround Challenges of Cost Bioinformatics Time Interpretation Analysis Primary BioWatch assay Most inclusive X X X X Environmental and detection Enable detection of "natural" positive filter background X X X controls (fungal spores, etc.) measurement Shotgun sequencing, Metagenomic Better understand sources of false random sequencing, sequencing positive signals unbiased sequencing Improve design of primers and probe targets used for primary screening Identification of novel or divergent Novel pathogen discovery pathogens that would go undetected X X X by existing tests Optimize design of primers Will identify new targets for PCR and Whole-genome X Microbial whole- and probes improve primer / probe design resequencing, de novo genome sequencing genome assembly Will better capture target diversity and Expand reference databases X X inform primer / probe design Investigate false-positive Better understand sources of false Amplicon sequencing, X X X results positive signals biased sequencing, Targeted sequencing conserved region More informative than "yes-no" result sequencing Primary BioWatch assay from real-time PCR; more inclusive of X X diverse strains 135
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From page 136... ...
In the nearer term, targeted approaches coupled with NGS may be useful to the program as a replacement for the current real-time PCR secondary assays because of their ability to analyze many more genomic regions for identification and characterization. The applicability of metagenomic NGS to the program would be a longer-term prospect.
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