Duplex Quantitative PCR Assay Research Articles

Simultaneous detection and differentiation of porcine circovirus 3 and 4 using a SYBR Green І-based duplex quantitative PCR assay

Porcine circovirus 4 (PCV4) was a novel circovirus identified from diseased pigs in 2019 in Hunan Province, China, and PCV3 and PCV4 co-infection has been reported. In order to detect and differentiate PCV3 and PCV4 simultaneously, the SYBR Green І-based duplex quantitative PCR (qPCR) assay was established in the present study. The two viruses could be easily distinguished by different Tm values: 86.5°C for PCV3 and 79°C for PCV4, while other porcine pathogens did not shown specific melting peaks. The detection limits of this duplex qPCR assay were 51.7 copies/μL for PCV3 and 67.7 copies/μL for PCV4, and both of the intra-assay and inter-assay of the CV analysis of this assay were less than 2.0 %. Sixty-four clinical samples from 22 different swine farms were screened by the duplex qPCR assay. The results showed that the positive detection rate of PCV3 was 37.5 % (24/64) and PCV4 was 34.38 % (22/64), and PCV3 and PCV4 co-infection rate was 17.19 % (11/64). The detection rate of the duplex qPCR assay was higher than that of the conventional PCR assay. The duplex qPCR was of high sensitivity and specificity, being able to provide technical support for clinical detection, differential diagnosis and control of PCV3 and PCV4.

A duplex SYBR Green I real-time quantitative PCR assay for detecting Escherichia coli O157:H7

PCR and hybridization assays are widely used for the detection and identification of Escherichia coli serogroups and serotypes. We used these techniques for the detection of E. coli O157:H7, a dominant serogroup among E. coli strains that are considered major public health problems worldwide. We developed a quantitative PCR assay using SYBR Green I, based on the published sequences of the rfbE and fliC genes from E. coli O157:H7. This method detected the E. coli O157:H7 O somatic antigen gene and the flagellar antigen gene simultaneously, with good specificity, sensitivity, and repeatability. The sensitivity of the assay was 2.95 x 10 copies/µL, which is 10(3) times more sensitive than obtained with a conventional PCR. The intra-assay and inter-assay coefficients of variation were less than 2%. We concluded that this duplex quantitative PCR assay is adequate for the identification and quantitative analysis of E. coli O157:H7. This provides a new identification method for clinical diagnosis of E. coli O157:H7 and for food safety analysis, as well as for molecular epidemiological studies of foodborne diseases.