摘要
为建立快速准确的弓形虫和钩端螺旋体检测方法,以弓形虫529 bp重复序列和钩端螺旋体LigB基因保守区域为靶标分别设计特异性引物和TaqMan MGB探针,进行双重荧光定量PCR反应体系和条件优化以及敏感性、特异性、重复性试验和临床样品检测。结果显示,该方法对弓形虫核酸检测限为1 copy/μL,钩端螺旋体核酸检测限为10 copies/μL;可特异性检测弓形虫和钩端螺旋体,与犬细小病毒、犬腺病毒II型、猫疱疹病毒、隐孢子虫、猫细小病毒等病原核酸无交叉反应;组内变异系数小于3.16%,组间的变异系数小于2.23%;临床样品检测验证该方法阳性检出率高于普通PCR方法,两种方法具有良好一致性。结果表明,所建立的双重荧光定量PCR方法具有敏感、特异、稳定等特性,可用于弓形虫和钩端螺旋体临床检测。
In order to develop a rapid and accurate method for detecting Toxoplasma gondii(T.gondii)and Leptospira,specific primers and TaqMan MGB probes were designed targeting 529 bp repetitive sequence of T.gondii and conserved region of LigB gene of Leptospira,respectively,and optimized in terms of qPCR reaction system and conditions,followed by testing of its sensitivity,specificity and repeatability,as well as detection of clinical samples.The results showed that the detection limit was 1 copy/μL for T.gondii nucleic acid,and 10 copies/μL for Leptospira nucleic acid;T.gondii and Leptospira were specifically detected,other than nucleic acids from pathogens such as canine parvovirus,canine adenovirus type 2,feline herpesvirus,Cryptosporidium and feline parvovirus;the intra-and inter-assay coefficients of variability was below 3.16%and 2.23%,respectively;for detection of clinical samples,the positive detection rate was higher than that by conventional PCR,from which good conformance was observed.In conclusion,the established method,characterized by high sensitivity,specificity and stability,could be used for the clinical detection of T.gondii and Leptospira.
作者
栾扬
张亚
李志敏
王蕾
毕振威
钱晶
熊富强
谭业平
Luan Yang;Zhang Ya;Li Zhimin;Wang Lei;Bi Zhenwei;Qian Jing;Xiong Fuqiang;Tan Yeping(Xizang Agriculture and Animal Husbandry College,Linzhi 860000,Xizang,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Key Laboratory of Veterinary Biologics Engineering and Technology,Ministry of Agriculture and Rural Aff airs,Nanjing 210014,Jiangsu,China;Nantong Egens Biotechnology Co.,Ltd.,Nantong 226010,Jiangsu,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,Jiangsu,China)
出处
《中国动物检疫》
2026年第1期118-123,130,共7页
China Animal Health Inspection
基金
江苏省农业自主创新项目[cx(24)3074]。
