牛支原体双重PCR检测方法的建立  

Establishment of a duplex-PCR assay for detecttion of Mycoplasma bovis

作　　者：刘洋[1] 李媛[1] 董惠[2] 张美晶[1] 姜海芳[1] 陈维[2] 辛九庆[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/动物细菌病研究室/国家牛传染性胸膜肺炎指定检测实验室,黑龙江哈尔滨150001 [2]东北农业大学动物医学学院,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2010年第8期599-602,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：兽医生物技术国家重点实验室基本科研业务费(NKLVBP200809)

摘　　要：为鉴别牛支原体(Mycoplasma bovis)与丝状支原体丝状亚种SC(M.mycoides subsp.mycoides SC),本实验通过优化M.bovis特异性引物pMB81-1/pMB81-2s和MmmSC特异性引物SC1/SC2的退火温度,建立了鉴别M.bovis和MmmSC的双重PCR检测方法。该方法能够分别由M.bovis和MmmSC扩增得到528bp和270bp片段。敏感性试验结果显示该方法检测M.bovis和MmmSC培养物的最低浓度分别为106cfu/mL和105cfu/mL。特异性试验结果显示,该方法对无乳支原体代表株PG2、丝状支原体丝状亚种LC型代表株Y-goat、山羊支原体山羊肺炎亚种Mccp、绵羊支原体Y-98、猪鼻支原体BST-7、巴氏杆菌以及结核分枝杆菌扩增结果均为阴性。应用该方法对临床病料的检测结果与培养鉴定结果的符合率为100%,表明该方法具有良好的特异性和敏感性,可以应用于临床检测。Mycoplasma bovis is a primary agent of respiratory disease which has similar sydromes and pathology characters to the contagious bovine pleuropneumonia caused by M. mycoides subsp. mycoides SC （MmmSC）. In this study, a duplex-PCR assay was established for the differential detection of M. bovis and MmmSC using primers specific to M. bovis and MmmSC, repectively. The duplex-PCR had a detection limit of 106 cfu/mL for M. bovis and 105 cfu/mL for MmmSC. It specifically amplified a DNA fragment of 528 bp from the M. bovis and 270 bp from the MmmSC type strain PG1, and no PCR products were amplified from the Mycoplasma agalactiae type strain PG2, Mycoplasma mycoides subsp. mycoides LC type strain Y-goat, M. capricolum subsp. Capripneumoniae, M. ovipneumonia type strain Y-98, M. hyorhinis, Pasteurella multocida and Mycobacterium. The method was applied to detect clinical samples and the results showed 100 % consistence with those of the bactera culture test.

关 键 词：牛支原体 丝状支原体丝状亚种SC 双重PCR 

分 类 号：S852.6[农业科学—基础兽医学]