Table_1_Development of a triplex quantitative reverse transcription-polymerase chain reaction for the detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus A.docx

Porcine viral diarrhea is caused by many pathogens and can result in watery diarrhea, dehydration and death. Various detection methods, such as polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR), have been widely used for molecular diagnosis. We developed a triplex real-time quantitative reverse transcription PCR (qRT-PCR) for the simultaneous detection of three RNA viruses potentially associated with porcine viral diarrhea: porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus A (PoRVA). The triplex qRT-PCR had R2 values of 0.999 for the standard curves of PEDV, TGEV and PoRVA. Importantly, the limits of detection for PEDV, TGEV and PoRVA were 10 copies/μL. The specificity test showed that the triplex qRT-PCR detected these three pathogens specifically, without cross-reaction with other pathogens. In addition, the approach had good repeatability and reproducibility, with intra-and inter-assay coefficients of variation <1%. Finally, this approach was evaluated for its practicality in the field using 256 anal swab samples. The positive rates of PEDV, TGEV and PoRVA were 2.73% (7/256), 3.91% (10/256) and 19.14% (49/256), respectively. The co-infection rate of two or more pathogens was 2.73% (7/256). The new triplex qRT-PCR was compared with the triplex RT-PCR recommended by the Chinese national standard (GB/T 36871-2018) and showed 100% agreement for PEDV and TGEV and 95.70% for PoRVA. Therefore, the triplex qRT-PCR provided an accurate and sensitive method for identifying three potential RNA viruses for porcine viral diarrhea that could be applied to diagnosis, surveillance and epidemiological investigation.

猪病毒性腹泻（Porcine viral diarrhea）由多种病原体引发，可导致水样腹泻、脱水甚至死亡。目前，聚合酶链式反应（polymerase chain reaction, PCR）、实时荧光定量聚合酶链式反应（real-time quantitative PCR, qPCR）等多种检测方法已被广泛应用于分子诊断。本研究开发了一种三重实时荧光定量反转录聚合酶链式反应（triplex real-time quantitative reverse transcription PCR, qRT-PCR），用于同时检测三种可能与猪病毒性腹泻相关的RNA病毒：猪流行性腹泻病毒（porcine epidemic diarrhea virus, PEDV）、猪传染性胃肠炎病毒（porcine transmissible gastroenteritis virus, TGEV）以及A群猪轮状病毒（porcine rotavirus A, PoRVA）。该三重qRT-PCR针对PEDV、TGEV和PoRVA的标准曲线决定系数（R²）均为0.999。尤为重要的是，该方法对PEDV、TGEV和PoRVA的检测限均为10拷贝/微升。特异性试验结果表明，该三重qRT-PCR可特异性识别这三种病原体，与其他病原体无交叉反应。此外，该方法具有良好的重复性与复现性，批内及批间变异系数均小于1%。最后，本研究使用256份肛门拭子样本对该方法的现场实用性进行了评估。PEDV、TGEV及PoRVA的阳性率分别为2.73%（7/256）、3.91%（10/256）及19.14%（49/256）。两种及以上病原体的混合感染率为2.73%（7/256）。本研究将该新型三重qRT-PCR与中国国家标准（GB/T 36871-2018）推荐的三重反转录聚合酶链式反应（triplex RT-PCR）进行对比，结果显示其对PEDV和TGEV的检测符合率为100%，对PoRVA的检测符合率为95.70%。综上，该三重qRT-PCR为猪病毒性腹泻相关的三种潜在RNA病毒提供了一种精准且灵敏的检测方法，可应用于临床诊断、疫情监测及流行病学调查。