Establishment and Application of a Duplex qPCR for NADC30-like and NADC34-like PRRSV
In recent years,NADC30-like and NADC34-like porcine reproductive and respiratory syndrome viruses(PRRSV)have caused serious economic loss to pig industry.In order to simultaneously detect NADC30-like and NADC34-like PRRSV,primers and probes were designed based on the region with obvious difference of ORF5 genes of classical strains,highly pathogenic strains,NADC30-like and NADC34-like strains.The concentration of primers and probes as well as annealing temperature were optimized to establish a duplex qPCR for NADC30-like and NADC34-like PRRSV,followed by evaluation of its specificity,sensitivity and repeatability to compare the results of clinical samples by the established method with those by the method reported in documentations.The results showed that the established method was positive only for the amplification of NADC30-like and NADC34-like PRRSV nucleic acids,but failed for other common pig pathogens,and no cross-reaction was found between the detection for NADC30-like and NADC34-like PRRSV;the minimum detection limits for NADC30-like and NADC34-like PRRSV were 1 and 10 copies/μL,respectively,and the intra-and inter-group coefficients of variation were both less than 2％;the results by the established method for NADC30-like and NADC34-like PRRSV were 89.5％and 92.9％coincident with those by the method reported in documentations,respectively.In conclusion,the duplex qPCR established in this study could be used to effectively distinguish NADC30-like from NADC34-like PRRSV with its advantages of high specificity,sensitivity and stability,supporting the diagnosis and epidemiological investigation for the disease.
NADC30-like PRRSVNADC34-like PRRSVduplex qPCRestablishment and application
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