摘要
为建立猫冠状病毒(feline coronavirus,FCoV)和猫细小病毒(feline parvovirus,FPV)的双重荧光定量PCR方法,选取FCoV 3'UTR和FPV VP2 基因保守区域,分别设计 2 对特异性引物和TaqMan MGB探针,并进行反应体系和条件优化,以及特异性、敏感性和重复性试验,探讨所建方法的可行性.结果显示:该方法可特异性检出FCoV和FPV,与猫疱疹病毒、猫杯状病毒、猫轮状病毒、支原体、衣原体、波氏杆菌、犬腺病毒和犬副流感病毒等病原核酸无交叉反应,对FCoV和FPV检测限均为1 copies/μL;FCoV和FPV阳性参考质粒组间和组内重复试验变异系数均小于3%;对35份临床样本进行检测,发现建立的双重荧光定量PCR方法阳性检出率比普通PCR方法高.结果表明,本研究建立的双重荧光定量PCR方法具有灵敏、特异和稳定等优点,可用于临床FCoV和FPV感染的早期鉴别诊断.
Abstract
In order to develop a duplex fluorescent quantitative PCR assay to detect feline coronavirus(FCoV)and feline parvovirus(FPV),the conservative regions of FCoV 3'UTR and FPV VP2 gene were selected to design two pairs of specific primers and TaqMan-MGB probes,respectively,followed by optimization of the reaction system and conditions.Then the sensitivity,specificity and repeatability were tested to explore the feasibility of the established assay.The results revealed that FCoV and FPV could be specifically detected by the assay,while no cross-reaction with feline herpesvirus,feline calici virus,feline rotavirus,Mycoplasma,Chlamydia,Bordetella,canine adenovirus and canine parainfluenza virus,was found.The detection limits for both FCoV and FPV were 1 copies/μL;the variable coefficients of inter-group and intra-group repeated tests of FCoV and FPV positive reference plasmids were both less than 3%;and it was found that,through detection of 35 clinical samples,the positive rate detected by the established method was higher than that by the traditional PCR.In conclusion,the established method,with the advantages of high sensitivity,good specificity and stability,could be used for early differential diagnosis of clinical infection with FCoV and FPV.
国家科技期刊平台
NSTL
维普
万方数据