Establishment and application of a quadruple target real-time PCR assay for detecting Yersinia pestis.

Abstract

In order to establish an efficient method for high-throughput detection of Yersinia pestis (Y. pestis), a quadruple target real-time polymerase chain reaction assay was developed based on the specific target genes of Y. pestis (caf1, pla, ymt, and ypo-1094). Its sensitivity, specificity, and repeatability were evaluated, and clinical serum samples were tested by the established method. The results showed that no cross-reactivity was observed with other bacterial nucleic acids. The optimal linear detection range for caf1, pla, ymt, and ypo-1094 was 12.09 × 10-⁶-12.09 × 101 ng/μL, and the lower limit of detection was 12.09 × 10-4 ng/μL. Four different DNA concentrations of caf1, pla, ymt, and ypo-1094 (10-4, 10-3, 10-2, 10-1and 101 ng/μL) were tested five times, achieving good repeatability. In the clinical sample detection, all the Y. pestis - positive samples were identified. The established method has potential for clinical use for rapid detection of Y. pestis with high specificity and high sensitivity.