中国兽医科学2018，Vol.48Issue(6)：692-699,8.DOI:10.16656/j.issn.1673-4696.2018.0120
猪丁型冠状病毒TaqMan实时定量RT-PCR检测方法的建立和应用
Development and application of a real-time TaqMan fluorescent quantitative RT-PCRassay for detection of porcine deltacoronavirus
摘要
Abstract
Primers and TaqMan fluorescent probe were designed according to the conserved regions of Ngene sequences of porcine deltacoronavirus (PDCoV), a recombinant plasmid containing Ngene of PDCoV was constructed as the positive standard sample, then a real-time TaqMan fluorescent quantitative RT-PCR (FQ-RT-PCR) assay was developed by optimization of reacting conditions. Furthermore, the sensitivity, specificity and repetition assay of the FQ-RT-PCR were tested. The results showed that there was no cross-reaction with porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus group A, porcine kobuvirus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, classical swine fever virus and porcine parvovirus. The assay was linear for the template plasmid pMD18-PDCoV-Nin the range from 2. 2×109—2. 2×101 copies/ L, the standard equation was Ct=-3. 539×lgX+38. 95, correlation coefficient R2=1. 0, and detection limit was low to 2. 2 copies/ L. Also, the assay showed the excellent reproducibility with the coefficient of variation less than 1. 0％. Eighteen of 64 clinical fecal samples collected from different areas in Guangxi were positive for PDCoV tested by the assay developed in this study, and the positive rate was 28. 1％, indicating there was PDCoV infection in Guangxi swine herds. In conclusion, the development of this assay provides a rapid, sensitive and specific diagnostic method for PDCoV detection and quantitative analysis.关键词
猪丁型冠状病毒/荧光定量RT-PCR/TaqMan探针Key words
porcine deltacoronavirus/real-time TaqMan fluorescent quantitative PCR/TaqMan fluorescence probe分类
农业科技引用本文复制引用
秦毅斌,赵武,何苹萍,卢冰霞,韦嫔媛,何颖,李斌,苏乾莲,段群棚,周英宁..猪丁型冠状病毒TaqMan实时定量RT-PCR检测方法的建立和应用[J].中国兽医科学,2018,48(6):692-699,8.基金项目
广西创新驱动发展专项资金项目(桂科AA17204057) （桂科AA17204057）
广西自然科学基金项目(2017GXNSFBA198092) （2017GXNSFBA198092）
广西兽医生物技术重点实验室开放基金课题(16-380-45-B-3) （16-380-45-B-3）
广西水产畜牧科技项目(桂渔牧科201633041,201633044,201633034) （桂渔牧科201633041,201633044,201633034）
广西基本科研业务费专项(桂科专项16-2,17-2) （桂科专项16-2,17-2）
南宁市西乡塘区科技开发项目(2017-2-10310, 201710317) （2017-2-10310, 201710317）
