畜牧与兽医2023，Vol.55Issue(12)：55-61,7.
4种牛分枝杆菌特异性基因PCR检测方法的比较与评价
Comparative study and evaluation of PCR methods for identifying Mycobacterium bovis by four different specific genes
摘要
Abstract
Mycobacterium bovis is an important zoonotic pathogen that seriously harms the cattle industry.Polymerase chain reaction(PCR)is one of the effective methods to detect the pathogen.In our study,the best PCR detection method was selected to provide technical reference for clinical surveillance of Mycobacterium bovis infection.Four pairs of primers were designed for four specific genes(16S rRNA,IS6110,IS1081 and Mpb64)of Mycobacterium bovis,and the optimal annealing temperature was determined by temperature gradient PCR.The nucle-ic acid of the reference strain of Mycobacterium bovis was used to determine the minimum detection limit of PCR.At the same time,the Chi-nese reference strain of Mycobacterium bovis,the international reference strains of Mycobacterium bovis,Mycobacterium avium,intracellular mycobacterium,Mycobacterium paratuberculosis,Brucella ovis and Brucella bovis were used for specific comparative tests.Finally,the detec-tion effects of different PCR methods were compared using artificial tissue samples(lungs,lymph nodes,milk samples)infected with Myco-bacterium bovis.Four pairs of primers were able to detect Mycobacterium bovis when the annealing temperature was 60 ℃,and primers 2-4 specifically detected Mycobacterium bovis.Primers 2 and 3 showed the highest sensitivity in detecting the genome of Mycobacterium bovis,reaching 10-10 ng/μL.The sensitivity of primer 2 in detecting Mycobacterium bovis in the lungs and lymph nodes of the tested cattle was high-er than 106 CFU/mL,and the sensitivity of primer 4 in detecting Mycobacterium bovis in the milk samples from the cattle was as high as 102 CFU/mL.In this study,the most appropriate PCR detection method was selected,providing reference for pathogenic detection of bovine tuberculosis and its application in clinical practice.关键词
牛分枝杆菌/病原/PCR/灵敏度/特异性Key words
Mycobacterium bovis/pathogen/PCR/sensitivity/specificity分类
农业科技引用本文复制引用
辛凌翔,王豪杰,刘燕,姚文生,胡云皓,张一帜,赵浩然,鑫婷,朱良全..4种牛分枝杆菌特异性基因PCR检测方法的比较与评价[J].畜牧与兽医,2023,55(12):55-61,7.基金项目
国家重点研发计划项目(2022YFD1800703) （2022YFD1800703）
中国兽医药品监察所-公益性专项(GY202103) （GY202103）
