Development of a Real-Time TaqMan RT-PCR Assay for the Detection of NADC34-like Porcine Reproductive and Respiratory Syndrome Virus

SIMPLE SUMMARY: It is vitally important that scientists are able to describe their work simply and concisely to the public, especially in an open-access online journal. The simple summary consists of no more than 200 words in one paragraph and contains a clear statement of the problem addressed, the aims and objectives, pertinent results, conclusions from the study, and how they will be valuable to society. This should be written for a lay audience, i.e., no technical terms without explanations. No references are cited, and no abbreviations. Submissions without a simple summary will be returned directly. ABSTRACT: NADC34-like porcine reproductive and respiratory syndrome virus first appeared in 2017 in a herd of pigs in Liaoning Province, China. The virus was subsequently found in other provinces. Given the potential for this virus to cause an epidemic, rapid, sensitive, and specific detection of NADC34-like PRRSV is required. The virus’ ORF5 gene was artificially synthesized based on a Chinese reference strain, and specific primers/probes for the ORF5 gene were designed. Then, the amplified target fragment was cloned into the pMD19-T vector, and a series of diluted recombinant plasmids were used to generate a standard curve. An optimized real-time TaqMan RT-PCR method was established. The method was highly specific for NADC34-like PRRSV, without cross-reactions with other non-targeted pig viruses. The detection limit of this assay was 10(1) copies/μL. The method had an efficiency of 98.8%, a squared regression value (R(2)) of 0.999, and showed a linear range of 10(3)–10(8) copies/μL of DNA per reaction. This method was shown to be analytically specific and sensitive with a low intra- and inter-assay coefficient of variation (<1.40%). A total of 321 clinical samples were tested using the established method, and four were shown to be positive (1.24%). This study confirmed the existence of NADC34-like PRRSV and HP-PRRSV co-infection in Sichuan and provided a promising alternative tool for the rapid detection of NADC34-like PRRSV.