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Due to the occurrence of natural plague outbreaks and its historical usage as a biological weapon, Yersinia pestis is considered one of the high-priority biological threat agents. It can remain viable in certain environments including water for >100 days. Because of its slow-growth characteristic, it usually takes three or more days to detect and confirm the identity of viable Y. pestis cells by PCR, serological, or biochemical assays when using the traditional microbiological plate-culture-based analysis, and that too, assuming faster growing microbes present in a water sample do not mask the Y. pestis colonies and interfere with analysis. Therefore, a rapid-viability Polymerase Chain Reaction (RV-PCR) method was developed for detection of Y. pestis. The RV-PCR method combines 24 h-incubation broth culture in a 48-well plate, and pre- and post-incubation differential PCR analyses, thereby allowing for rapid and high-throughput sample analysis compared with the current plate culture method. One chromosomal and two plasmid gene target-based real-time PCR assays were down-selected, showing ca. 10 genome equivalent detection; the chromosomal assay was then used for RV-PCR method development. A 10-cell level (10-99 cells) sensitivity of detection was demonstrated even with complex sample backgrounds including known PCR inhibitors (ferrous sulfate and humic acid), as well as metal oxides and microbes present in Arizona Test Dust (ATD). The method sensitivity was maintained in the presence of dead Y. pestis cells up to 10 cells per sample. While affording high-throughput and rapid sample analysis, the 48-well plate format used in this method for sample enrichment significantly reduced labor requirements and generation of BioSafety Level-3 (BSL-3) laboratory waste as compared to the usual microbiological plate-culture-based methods. This method may serve as a model for other vegetative bacterial pathogens.
This article was published in the following journal.
Name: Journal of microbiological methods
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Methods for using more than one primer set in a polymerase chain reaction to amplify more than one segment of the target DNA sequence in a single reaction.
Methods used for detecting the amplified DNA products from the polymerase chain reaction as they accumulate instead of at the end of the reaction.
In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.
A heat stable DNA-DIRECTED DNA POLYMERASE from the bacteria Thermus aquaticus. It is widely used for the amplification of genes through the process of POLYMERASE CHAIN REACTION. EC 2.7.7.-.
A technique that labels specific sequences in whole chromosomes by in situ DNA chain elongation or PCR (polymerase chain reaction).
Polymerase Chain Reaction (PCR)
PCR (Polymerase Chain Reaction) uses the ability of DNA polymerase (enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to create two ident...
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