猪代尔塔冠状病毒荧光定量PCR检测方法的建立及其S基因分子特征分析

Establishment of one-step Taqman quantitative PCR detection method and molecular S gene characterization analysis of porcine deltacoronavirus

猪代尔塔冠状病毒(porcine deltacoronavirus,PDCo V)是一个新发现的致病性猪肠道冠状病毒,目前在中国部分省份养殖场的腹泻病猪中已检测到,并已证实在中国流行。本研究旨在探明PDCo V在浙江省的流行现状和分子演化特征。结果显示,建立的PDCo V一步法Taq Man探针荧光定量检测方法特异,检测灵敏度可达3.94×10~2拷贝·μL^(-1),扩增效率为108%,R^2值为0.997,标准曲线方程为Y=-3.156X+1.826。2013年4月—2016年12月收集的258份仔猪临床腹泻样本中没有检测到,但在2017年1—4月检测阳性率达到50%(12/24)。获得的7个临床分离株的S基因与参考株相比,其核苷酸和氨基酸的同源性分别为99.98%~100%和99.99%~100%;与最初在香港分离到的毒株和美国流行株相比,存在3个碱基缺失和一定数量的突变。以S蛋白进行分子演化分析,发现7株临床分离株均在代尔塔冠状病毒属进化分支上,且与中国分离到的其他PDCo V毒株在进化关系上较近,与美国、泰国等地分离到的PDCo V在进化关系上较远,提示PDCo V的流行株分布有一定的地域性。研究结果提供了浙江省地区PDCo V临床分离株的基因特性,为防控PDCo V引起的仔猪腹泻、加快流行株疫苗的开发提供了数据。

Porcine deltacoronavirus( PDCo V) is a newly discovered pathogenic porcine coronavirus that has been isolated from diarrheal pigs in farms of some provinces in China,confirming that the disease has been prevalent in our country. This study aims to investigate the prevalence and molecular evolution of PDCo V in Zhejiang Province. The results showed that the established PDCo V one-step Taq Man probe was specific for fluorescence quantitative detection. The detection sensitivity was 3. 94 × 10~2 copies·μL^(-1). The amplification efficiency was 108% and the R^2 val-ue was 0. 997,regression equation of standard curve is Y =-3. 156 X + 1. 826. The virus was not detected in clinical diarrhea samples of 258 piglests collected from April,2013 to December,2016,but the positive rate was 50%( 12/24) in the period from January to April,2017. The homology of the nucleotide and amino acid of the S gene of the seven clinical isolates was 99. 98%-100% and 99. 99%-100%,respectively. Compared with the reference strain and the Hongkong and American isolated trains,there were three base deletions and a number of mutations in our clinical isolated strains. S-protein was used for the molecular evolution analysis. Seven strains of clinical isolates were found to be belong to the evolutionary branch of the delta virus,and were closer to the other PDCo V strains isolated from China,while farther to the strains isolated from the United States and Thailand,suggesting that PDCo V epidemic strains have a certain geographical distribution. This study provides the genetic characteristics of PDCo V clinical isolates in Zhejiang Province,and provides data for the prevention and control of PDCo V-induced piglet diarrhea and the development of epidemic strains.