Establishment and application of a triplex RT-PCR method for detection of PDCoV, SADS-CoV, and SVA

ABSTRACT

[Background] Porcine delta coronavirus（PDCoV）, swine acute diarrhea syndrome coronavirus（SADS-CoV）, and Seneca virus A（SVA） are new pathogens which seriously endanger the development of pig industry. The clinical symptoms of pigs infected with the three pathogens are difficult to distinguish. Therefore, it is urgent to establish a multiplex RT-PCR detection method for rapid diagnosis of suspected pigs and reduce economic losses. [Objective] To establish a triplex RT-PCR method for simultaneous detection of single or mixed infection of PDCoV, SADS-CoV, and SVA. [Methods] Three pairs of specific primers were designed according to the conserved regions of the N genes of PDCoV and SADS-CoV and the L/P1 genes of SVA registered in GenBank, and the optimal annealing temperature（Tm） was determined by temperature gradient PCR method. The primer concentration was optimized by array method. The recombinant plasmids PMD-PDCoV, PMD-SADS-CoV,and PMD-SVA were constructed as standards to determine the limits of detection（LOD）. The specificity of the triplex RT-PCR method was determined with the nucleic acid samples of 6 common pig viruses including porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine reproductive and respiratory syndrome virus. The repeatability of the established method was verified by inter-batch and intra-batch tests. Finally, we employed the triplex RT-PCR method to detect the clinical samples suspected of infection and compared the results with those obtained with the reported detection methods, thus evaluating the clinical application performance of the method. [Results] The optimal Tm was 58.3 ℃, and the optimal primer concentrations were 0.5 μmol/L, 0.25 μmol/L, and 0.25 μmol/L,respectively. The established method had high sensitivity, with the LODs of 1 copy/μL, 1 copy/μL, and 10 copies/μL for PMD-PDCoV, PMD-SADS-CoV, and PMD-SVA, respectively. It had strong specificity, with specific bands only for PDCoV, SADS-CoV, and SVA and no bands for other viruses.Moreover, the method had good repeatability as the test results were consistent between and within batches. The positive rates of PDCoV, SADS-CoV, and SVA in the clinical samples detected by the established method were 65.85%, 30.49%, and 57.32%, respectively, which were consistent with the results obtained with the reported detection methods. Finally, 5 samples were randomly selected from 13 positive samples of PDCoV, SADS-CoV, and SVA for sequencing, and the phylogenetic tree indicated that the PCR amplification sequences of the five positive samples had high homology（above 96%） between each other and also with the reference sequences. [Conclusion] The triplex RT-PCR method established in this study is accurate and reliable for the simultaneous detection of PDCoV,SADS-CoV, and SVA in clinical samples.