Establishment and evaluation of internal control of multiplex quantitative PCR for detection of Yersinia pestis

  Objective  To establish an internal control of multiplex quantitative PCR for the detection of Yersinia pestis and evaluate its effect.

  Methods  A multiplex quantitative PCR system was established using the YPO0393 gene of the chromosome of Y. pestis and caf1 gene of plasmid pMT1 as the target gene. The internal control was YPO0393. Specific primers and probes were designed. Detection was performed for DNA of the control, Y. pestis and field samples. The specificity, sensitivity and repeatability of this assay were evaluated.

  Results  All the 8 DNA samples of Y. pestis at different concentrations showed significant S-curve amplification. The DNA of 12 controls showed no amplification curve, suggesting that the multiplex quantitative PCR was specific. The detection sensitivity of the multiplex quantitative PCR was 22.5×10⁻⁴ ng/μL, and the coefficient of variation ranged from 0.99 to 3.42 for the EV76 strain of Y. pestis, indicating good sensitivity and repeatability. In the 105 field samples, 6 were determined as positive by the multiplex quantitative PCR, which was consistent with routine PCR. Y. pestis was isolated from 4 positive field samples.

  Conclusion  The multiplex quantitative PCR has high specificity, sensitivity and repeatability by using the YPO0393 gene as internal control, by which the false negative rate can be reduced. Therefore, this assay can be applied in the rapid detection of Yersinia pestis.