哺乳动物正病毒(MRV)感染在包括人类在内的多种哺乳动物物种中普遍存在,主要引起胃肠炎和呼吸道疾病。在这项研究中,我们基于在保守的L1基因中设计的引物和探针,开发了一种快速灵敏的TaqManqRT-PCR检测MRV的方法。对qRT-PCR检测的灵敏度进行了评价,特异性,效率和重现性。发现检测灵敏度相当于10个DNA拷贝/μL,标准曲线的线性相关性为R2=0.998,扩增效率为99.6%。测定间和测定内的变异系数(CV%)在0.29%至2.16%和1.60%至3.60%的范围内,分别。引物组特异性地扩增了它们各自的MRV片段,并且具有最高的检测灵敏度,为100.25TCID50/mL,扩增效率为99.5%(R2=0.999)。qRT-PCR用于从绵羊样品中检测MRV,山羊,来自中国四个地区的小牛,整体MRV患病率为8.2%(35/429),而通过RT-PCR检测到17/429(4.0%),通过病毒分离检测到14/429(3.3%)。qRT-PCR分析显示出比RT-PCR和病毒分离显著更高的灵敏度。流行病学调查结果表明,陕西检测的绵羊和山羊直肠拭子中MRV的阳性率,江苏,新疆为9/80(11.3%),12/93(12.9%)和14/128(10.9%),分别。在山羊和绵羊中,MRV患病率与季节和年龄明显相关,在9月至4月期间阳性率超过8%,在两个月以下的小反刍动物中阳性率约为13%。这是中国绵羊和山羊MRV感染的首例,从而扩大了我们对MRV宿主的认识。因此,qRT-PCR的引物优化不仅应优先考虑扩增效率和特异性,还有敏感性。该方法将有助于通过流行病学调查更准确,快速地监测MRV,病毒载量,和疫苗接种功效。
Mammalian orthoreovirus (MRV) infections are ubiquitous in multiple mammalian species including humans, and mainly causes gastroenteritis and respiratory disease. In this study, we developed a rapid and sensitive TaqMan qRT-PCR method for MRV detection based on the primers and probe designed within the conserved L1 gene. The qRT-PCR assay was evaluated for its sensitivity, specificity, efficiency and reproducibility. It was found that the detection sensitivity was equivalent to 10 DNA copies/μL, and the standard curves had a linear correlation of R2 = 0.998 with an amplification efficiency of 99.6%. The inter- and intra-assay coefficients of variation (CV%) were in the range of 0.29% to 2.16% and 1.60% to 3.60%, respectively. The primer sets specifically amplified their respective MRV segments and had the highest detection sensitivities of 100.25 TCID50/mL with amplification efficiencies of 99.5% (R2 = 0.999). qRT-PCR was used for MRV detection from samples of sheep, goats, and calves from four regions in China, and the overall MRV prevalence was 8.2% (35/429), whereas 17/429 (4.0%) were detected by RT-PCR and 14/429 (3.3%) by virus isolation. The qRT-PCR assay showed significantly higher sensitivity than RT-PCR and virus isolation. Results from an epidemiological survey indicated that the positive rate of MRV in rectal swabs from sheep and goats tested in Shaanxi, Jiangsu, and Xinjiang were 9/80 (11.3%), 12/93 (12.9%) and 14/128 (10.9%), respectively. In goats and sheep, MRV prevalence was obviously associated with season and age, with a high positive rate of more than 8% during September to April and approximately 13% in small ruminant animals under two months of age. This is the first instance of MRV infection in sheep and goats in China, thus broadening our knowledge of MRV hosts. Consequently, primer optimization for qRT-PCR should not only prioritize amplification efficiency and specificity, but also sensitivity. This assay will contribute to more accurate and rapid MRV monitoring by epidemiological investigation, viral load, and vaccination efficacy.