检测绵羊源爱知病毒D型荧光RT-PCR方法的建立和应用

绵羊源爱知病毒D型(ovine Aichivirus D)是在国内绵羊中新发现的病毒，本研究根据绵羊源Aichivirus D 3D基因序列设计检测引物，通过反应体系和条件优化，成功建立了检测绵羊源Aichivirus D的TB Green染料法荧光RT-PCR方法，该方法特异性和稳定性良好，灵敏度高。对2020年4月―2021年5月采集自四川6个场253份绵羊粪便样本(健康羊的133份，腹泻羊的120份)进行检测，结果该病毒的平均检出率为8.7％，场阳性率为66.7％。其中，腹泻粪便样本中绵羊源Aichivirus D阳性率(17.5%)显著高于非腹泻粪便样本中绵羊源Aichivirus D阳性率(0.75%，P<0.001)。表明绵羊源Aichivirus D可能是引起以上地区绵羊腹泻的病原。从绵羊源Aichivirus D的阳性样本中成功克隆出大小为1 422 bp的13个完整的绵羊源Aichivirus D 3D基因，其核苷酸相似性为99.4％～100.0％。遗传演化分析发现这13株绵羊源Aichivirus D 3D基因与本实验室前期研究上传的绵羊源Aichivirus D 3D基因共同聚为单独的一个大支。本研究为绵羊源Aichivirus D的分子检测提供了一种新的方法和基础流行病学数据。

Establishment and Application of a Fluorescent RT-PCR Assay for Detecting Ovine Aichivirus D

Ovine Aichivirus D is an emerging virus in sheep in China, the aim of this study was to design primers targeting 3D gene of ovine Aichivirus D and optimize the reaction system and conditions, a TB Green Fluorescent RT-PCR method was successfully established; the assay has characteristics of good specificity and stability, high sensitivity. Total 253 sheep fecal samples (133 non-diarrheic and 120 diarrheic) were collected from 6 sheep farms in Sichuan province from April 2020 to May 2021, this virus detection rate was 8.7%, and the farm positive rate was 66.7%(4/6). Notably, the ovine Aichivirus D positive rate for the diarrheic feces (17.5%) was significantly higher than that for non-diarrheic feces (0.75%, P<0.001), suggesting that ovine Aichivirus D may be a pathogen of sheep diarrhea. In addition, 13 complete 3D genes in 1 422 bp in length were successfully cloned from ovine Aichivirus D positive samples in this study, which shared 99.4%-100.0% nt homology with each other. Evolutionary analysis showed that these 13 3D genes together with the ovine Aichivirus D 3D gene uploaded by our previous research clustered into a large independent branch. This study provides a new molecular tool for detecting ovine Aichivirus D and basic epidemiological data of ovine Aichivirus D.