Real‐time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis |
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| Authors: | PIRJO MATERO,TANJA PASANEN,RIIKKA LAUKKANEN,P IVI TISSARI,EVELIINA TARKKA,MARTTI VAARA,MIKAEL SKURNIK |
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| Affiliation: | 1. Helsinki University Central Hospital Laboratory Diagnostics, Helsinki;2. Centre of Military Medicine, Helsinki;3. Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Helsinki;4. Department of Bacteriology and Immunology, Infection Biology Research Program, Haartman Institute, University of Helsinki, Helsinki, Finland |
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| Abstract: | A multiplex real‐time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage λ; the latter was used as an internal amplification control. The Y. pestis‐specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10–100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively. |
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| Keywords: | Yersinia pestis Yersinia pseudotuberculosis real‐time PCR multiplex pla wzz ypo2088 |
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