Development and implementation of a TaqMan triplex real-time PCR assay for concurrent detection of pseudorabies virus, porcine teschovirus 1, and Streptococcus suis 2

Ranran Lai^1,2Chen Yang^1,2Xiaowen Li^1,2,3,4*Zhiqiang Hu^5*lili wu^1,2,4Weisheng Wu³Lulu Li²Wei Liu³Zheng Yan²Diankun Yu³Shengzhi Ren¹

• ¹Shandong Engineering Research Center of Pig and Poultry Health Breeding and Important Infectious Disease Purification, Shandong New Hope Liuhe Group Ltd, Qingdao, China
• ²Juye Xinhao Agriculture and Animal Husbandry Co., Ltd, Heze, China
• ³New Hope Liuhe Co.,Ltd./Key Laboratory of Feed and Livestock and Poultry Products Quality & Safety Control, Ministry of Agriculture, Chengdu, Sichuan Province, China
• ⁴China Agriculture Research System-Yangling Comprehensive Test Station, Yangling, China
• ⁵Key Laboratory of Animal Epidemic Disease Detection and Prevention in Panxi District, College of Animal Science, Xichang University, Xichang, China

Porcine neurological disorders represent a prevalent clinical condition that leads to significant mortality and economic losses within the swine industry. pseudorabies virus (PRV), porcine teschovirus 1 (PTV1), and Streptococcus suis 2 (SS2)are key viral and bacterial pathogens implicated in the manifestation of neurological symptoms in pig populations. The overlapping clinical presentations and pathological alterations associated with these pathogens pose challenges in their clinical differentiation.Therefore, it is essential to develop a diagnostic method with high sensitivity and specificity that can simultaneously detect and differentiate these viral and bacterial agents.A triplex real-time PCR assay using TaqMan probes was developed to simultaneously detect PRV, PTV1, and SS2. To assess the efficacy of the established assay, 30 clinical samples of animals with nervous symptoms were used to compare the results obtained from the triplex real-time PCR assay with those obtained from commercial singleplex real-time PCR kits. Furthermore, a total of 282 samples were tested and analyzed to validate the utility of the assay.The triplex real-time PCR assay exhibited high sensitivity, specificity, and repeatability, with a detection limit of 1.0 × 10 0 copy/μL. The triplex real-time PCR method and commercial singleplex real-time PCR kits showed complte concordance in detecting PRV, PTV1, and SS2. Clinical data indicated single infection rates of 8.16% for PRV, 26.95% for PTV1, and 7.80% for SS2. The observed co-infection rates were 7.45% for PRV+PTV1, 0.71% for PRV+SS2, 1.42% for PTV1+SS2, and 1.77% for PRV+PTV1+SS2, respectively.The triplex real-time PCR method developed in this study effectively distinguishes PRV, PTV1, and SS2 simultaneously, serving as a valuable diagnostic tool. This method is anticipated to play a crucial role in preventing and controlling infectious disease spread and supporting epidemiological investigations.