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

牛分枝杆菌是严重危害养牛业的重要人兽共患病原微生物,PCR是检测该病原的有效手段之一。本研究通过筛选最佳的PCR检测方法,以期为临床监测牛分枝杆菌感染提供技术参考。针对牛分枝杆菌4种常见的特异性基因(16S rRNA、IS6110、IS1081和Mpb64)分别设计引物,通过温度梯度PCR法确定最适退火温度;采用牛分枝杆菌参考株核酸确定PCR最小检测限,同时运用牛分枝杆菌国内参考株、牛分枝杆菌国际参考株、禽分枝杆菌、胞内分枝杆菌、副结核分枝杆菌、羊种布鲁菌以及牛种布鲁菌进行特异性比较试验;最后运用人工模拟牛分枝杆菌感染组织样本(肺脏、淋巴结、奶样)比较不同PCR方法检测效果。4对引物以60℃为退火温度时均能检测分枝杆菌,其中IS6110、IS1081和Mpb64基因引物可以特异性检测牛分枝杆菌。IS6110和IS1081基因引物对牛分枝杆菌基因组的检测灵敏度最高,可达10^(-10) ng/μL;IS6110基因引物在肺脏和淋巴结中牛分枝杆菌检测灵敏度可达10~6 CFU/mL以上,而Mpb64基因引物对奶样中牛分枝杆菌的检测灵敏度可达10~2 CFU/mL。本研究筛选出相对适宜的牛分枝杆菌PCR检测方法,为牛结核病病原学检测和临床应用提供技术支持。

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 nucleic acid of the reference strain of Mycobacterium bovis was used to determine the minimum detection limit of PCR.At the same time,the Chinese 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 detection effects of different PCR methods were compared using artificial tissue samples(lungs,lymph nodes,milk samples) infected with Mycobacterium 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 higher than 10~6 CFU/mL,and the sensitivity of primer 4 in detecting Mycobacterium bovis in the milk samples from the cattle was as high as 10~2CFU/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.