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. 1998 Aug;36(8):2284-8.
doi: 10.1128/JCM.36.8.2284-2288.1998.

5' nuclease PCR assay to detect Yersinia pestis

Affiliations

5' nuclease PCR assay to detect Yersinia pestis

J A Higgins et al. J Clin Microbiol. 1998 Aug.

Abstract

The 5' nuclease PCR assay uses a fluorescently labeled oligonucleotide probe (TaqMan) to rapidly detect and quantitate DNA templates in clinical samples. We developed a 5' nuclease PCR assay targeting the plasminogen activator gene (pla) of Yersinia pestis. The assay is species specific, with a detection threshold of 2.1 x 10(5) copies of the pla target or 1.6 pg of total cell DNA. The assay detected Y. pestis in experimentally infected Xenopsylla cheopis fleas and in experimentally infected monkey blood and oropharyngeal swabs. The TaqMan assay is simple to perform and rapid and shows promise as a future field-adaptable technique.

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Figures

FIG. 1
FIG. 1
Sensitivity of the TaqMan assay for Y. pestis. Total (chromosomal and plasmid) DNA isolated from Y. pestis K25 cells was used as the template in quantities of 5, 1, 0.2, 0.04, 0.008, and 0.0016 ng. Primers for a 344-bp region of the Y. pestis pla gene were used with 10 pmol of the MS002 probe per sample. The graph depicts amplification plots for each template; plot 1 is the 5-ng template, etc. Plot 7 is a no-template control. Inset: agarose gel electrophoresis image of respective PCR products. Lane M, 100-bp DNA ladder. Lane 1 corresponds to plot 1, etc. Lane 7 is a no-template control. (This figure was previously presented as a poster at a Department of Defense meeting.)
FIG. 2
FIG. 2
Results of TaqMan assays performed on oropharyngeal swabs from monkeys experimentally aerosol infected with Y. pestis. The assay used pla primers and 10 pmol of the MS002 probe per sample. (A) Graph depicting the amplification plots for three monkeys (plots 1 to 3) and two no-template controls (plots 4 and 5; for clarity, positive control amplification plots have been omitted). (B) Agarose gel electrophoresis image of pla amplification products. Lanes: 4 and 5, no-template and extraction controls, respectively; 1 to 3, monkey samples; M, low-mass DNA ladder. (C) Agarose gel electrophoresis image of β-globin primer amplification products. Lanes: 6, human DNA positive control; 5, contamination control; 4, extraction control; 1 to 3, monkey samples; M, low-mass DNA ladder.
FIG. 3
FIG. 3
Results of Y. pestis TaqMan assays performed on infected X. cheopis fleas with Y. pestis pla primers and 10 pmol of the MS002 probe per sample. The graph depicts the amplification plots for eight fleas (plots 1 to 8) and a no-template control (plot 9) (for clarity, positive control amplification plots have been omitted). The inset is an agarose gel electrophoresis image of the amplification products from fleas (samples 1 to 8) and a no-template control (sample 9). Lane M is a low-mass DNA ladder. Lane pos is a Y. pestis DNA positive control.

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