山羊支原体山羊肺炎亚种特异性分子检测靶标的鉴定和应用

Identification and application of a specific molecular target for detection of Mycoplasma capricolum subsp.capripneumoniae

本文旨在筛选出新的山羊支原体山羊肺炎亚种(Mycoplasma capricolum subsp.capripneumoniae,Mccp)特异性分子诊断靶标。首先通过全基因组本地BLASTp方法筛选得到Mccp理论上特异的蛋白基因序列,经BLASTn验证、优选特异性基因92.1作为候选靶标,设计引物92.1F/R,采用普通PCR体系检测4株Mccp(1801、1601、1309F、Zly-14)和7种其他支原体基因组DNA,包括无乳支原体(Ma)PG2、丝状支原体山羊亚种(Mmc)PG3、Mmc YG(前称丝状支原体丝状亚种大菌落型,MmmLC)、绵羊肺炎支原体(Mo)Y98、山羊支原体山羊亚种(Mcc)Ckid、牛支原体(Mb)08M、李奇氏支原体(Ml)PG50,并检测Mccp、Mo、Mmc人工感染羊试验样本DNA,评估该体系的特异性。结果表明92.1F/R引物只能从Mccp菌株及其感染试验样本中扩增出509 bp目的条带,证实该引物可用于检测Mccp,92.1序列可作为CCPP新的核酸诊断靶标,为CCPP诊断和实验室研究提供了一种新的分子标记。

To screen novel specific molecular target for Mycoplasma capricolum subsp.capripneumoniae(Mccp).We firstly got theoretical unique genes of Mccp with BLASTp and BLASTn tools,the specific gene sequence 92.1 was selected and used to design the primers 92.1 F/R,and the specificity of the primers was assessed by the PCR system with following DNA samples,Mccp strains 1801,Zly-14,1601 and1309 F,M.agalactiae(Ma)strain PG2,M.mycoides subsp.capri(Mmc)strain PG3,Mmc strain YG(formerly known as Mmm LC),M.ovipneumoniae(Mo)strain Y98,M.capricolum subsp.capricolum(Mcc)strain Ckid,M.bovis(Mb)strain 08 M,and M.leachi(Ml)strain PG50,as well as the recovered strains and samples from experimentally infected goats with Mccp,Mo and Mmc,respectively.In PCR,the primer set of 92.1 F/R was only able to amplify a 509 bp of targeted fragment from DNAs from Mccp strains and Mccp infected goats’samples.It indicates that the 92.1 primers set is specific to Mccp and the 92.1 gene sequence can be used as a novel molecular diagnostic target for Mccp,which provide a new molecular marker for CCPP diagnosis and research.