猪丁型冠状病毒荧光定量RT-PCR与S1蛋白间接ELISA检测方法的建立及应用

Development and application of real-time RT-PCR and S1 protein-based indirect ELISA for porcine deltacoronavirus

摘要:

最近，新发现的猪丁型冠状病毒 (Porcine deltacoronavirus，PDCoV) 可导致仔猪腹泻，亟待建立有效准确的核酸及血清学检测方法并调查猪场的PDCoV感染情况。本研究采用针对病毒M基因的探针荧光定量RT-PCR检测方法，对2014?2015年间收集的河南、湖南、浙江、江西、安徽、河北、黑龙江、江苏、山东和上海等10个省市收集的共254份腹泻仔猪小肠匀浆或粪便样本进行检测，共检出11份PDCoV阳性样本，总阳性率为4.33%。采用在昆虫细胞表达纯化的PDCoV囊膜S1蛋白为包被抗原，建立间接酶联免疫吸附试验 (ELISA) 方法，检测2015?2016年间收集的来自江苏、湖南、浙江等7个省份的609份具有腹泻症状的猪血清样品，抗PDCoV S1抗体检出率为44.17% (269/609)。上述建立的两种方法分别针对病毒的RNA或抗体对PDCoV进行特异性检测，可以应用于PDCoV流行情况的监控。

Abstract:

Porcine deltacoronavirus (PDCoV) has been recently recognized as an emerging viral pathogen that causes diarrhea in newborn piglets. A total of 254 small intestinal or fecal samples collected from 10 provinces including Henan, Hunan, Zhejiang, Jiangxi, Anhui, Hebei, Heilongjiang, Jiangsu, Shandong and Shanghai between 2014 and 2015, were screened by quantitative RT-PCR targeting the viral M gene. Eleven PDCoV positive samples were identified with a total positive rate of 4.33%. An indirect enzyme-linked immunosorbent assay (ELISA) was developed based on the recombinant S1 protein of PDCoV. This assay was used to test 609 serum samples of pigs with diarrhea symptoms collected from 10 provinces between 2015 and 2016. The positive rate of PDCoV antibody was 44.17% (269/609). The two methods can be used to monitor the PDCoV epidemiology in the levels of PDCoV specific RNA or antibody, helping better prevent and control PDCoV.