山羊伪结核棒状杆菌TaqMan荧光定量PCR检测方法的建立和初步应用  

Establishment and preliminary application of TaqMan real-time PCR for detection of Corynebacterium pseudotuberculosis in goats

作　　者：马玉馨 李文贵[1] 郑国英 邬培昆 邵庆勇 李志明 高华峰[2] MA Yuxin;LI Wengui;ZHENG Guoying;WU Peikun;SHAO Qingyong;LI Zhiming;GAO Huafeng(Yunnan Agricultural University,Kunming 650201,China;Yunnan Province Key Laboratory of Tropical and Subtropical Animal Virus Diseases,Kunming 650024,China;Animal Husbandry and Veterinary Station of Sishan District,Kunming 650101,China)

机构地区：[1]云南农业大学,云南昆明650201 [2]云南省热带亚热带动物病毒病重点实验室,云南昆明650024 [3]昆明市西山区畜牧兽医站,云南昆明650101

出　　处：《畜牧与兽医》2018年第10期82-87,共6页Animal Husbandry & Veterinary Medicine

基　　金：昆明市西山区2016年度科技项目;国家现代肉羊产业技术体系项目(CARS-39)

摘　　要：为了快速检测无临床症状的山羊是否感染山羊伪结核棒状杆菌（Corynebacterium pseudotuberculosis）,根据Gen Bank中伪结核棒状杆菌磷脂酶D（phospholipase D,PLD）基因序列,分别设计1对特异性引物和Taq Man探针,经过反应条件和体系优化,测定特异性、敏感性和稳定性,建立山羊伪结核病Taq Man荧光定量PCR检测方法,并对54份临床样品进行检测。结果显示：建立的山羊伪结核棒状杆菌方法具有很好的特异性,在20μL扩增体系中,引物浓度为0.20μmol/m L,探针浓度为0.45μmol/m L,退火温度59℃时最佳;最低可检测出4.6×102个拷贝数的细菌DNA,标准曲线相关系数是0.996。同时,对Taq Man荧光定量PCR和常规PCR进行了比较,前者的敏感性是后者的1 000倍;对54份临床样品进行检测,Taq Man荧光定量PCR检出的阳性率为68.5%（37/54）,常规PCR检出的阳性率为53.7%（29/54）。研究表明：Taq Man荧光定量PCR法检出率较高,有特异、敏感且快速的特点,适用于临床样品的检测。To quickly detect whether goats without clinical symptoms are infected with Corynebacterium pseudotuberculosis,to establish a Taq Man-based real-time PCR method for detection of C. pseudotuberculosis,primers and the Taq Man probe were designed based on the PLD genes of C. pseudotuberculosis in Genbank. The experimental reactive conditions of the real-time PCR method were optimized to improve its specificity,stability and sensitivity,and the standard curve was established. And detection of the infection was performed in 54 clinical samples. The results showed that the established Taq-Man real-time PCR had a high specificity. In the 20 μL amplification system,the best primer concentration was 0. 20 μmol/m L,the probe concentration was 0. 45 μmol/m L and the annealing temperature was 60℃. The detected limit was 4. 6×102copies of bacterial DNA,and the correlation coefficient of standard curve was 0. 996. The sensitivity of the real-time PCR was 103 times higher than that of PCR. Detection of 54 clinical samples by using the real-time PCR and PCR methods showed that the positive rate of these two methods were 68. 5%（ 37/54） and 53. 7%（ 29/54）,respectively. These results indicated the real-time PCR was more specific,sensitive and rapid than PCR,and was more applicable to clinical sample detection.

关 键 词：伪结核棒状杆菌 磷脂酶D基因(PLD) 实时荧光定量PCR 

分 类 号：S855.1[农业科学—临床兽医学]