山羊支原体山羊肺炎亚种可视化实时荧光LAMP检测方法的建立及应用

Development and application of visual and real-time fluorescent LAMPs for detection of Mycoplasmacapricolum subsp.capripneumoniae

为建立快速简便、灵敏度高、特异性强的山羊支原体山羊肺炎亚种(Mccp)核酸快速检测方法,选择Mccp的92.1基因序列为靶序列,设计2条外引物、2条内引物和2条环引物,并向反应体系中添加可视化染料羟基溴酚蓝(HNB)或荧光染料(Eva green),分别建立Mccp可视化环介导恒温扩增(V-LAMP)和实时荧光环介导恒温扩增(RF-LAMP)检测方法,并对比两种方法的敏感性、特异性、重复性以及两种方法在山羊组织样品中的检测效果。结果显示,两种方法均可检出1.0×10 copies/μL的核酸标准品,灵敏度高;与其他支原体及细菌,如丝状支原体山羊亚种、绵羊肺炎支原体、多杀性巴氏杆菌、肺炎克雷伯菌等类症病原体,均无交叉反应;V-LAMP、RF-LAMP的变异系数均小于1%;对人工感染的试验样品和临床样品进行检测,两种检测方法结果一致,阳性率为9.09%,并且与实时定量PCR检测结果一致。本试验建立的V-LAMP和RF-LAMP为Mccp流行病学调查和疾病诊断提供了新的临床快速检测方法。

In order to develop rapid,simple,highly-sensitive and specific loop-mediated isothermal amplification(LAMP) assays for detection of Mycoplasma capricolum subsp.capripneumoniae(Mccp),a set of primers including outer primers,inner primers and loop primers was designed based on the nucleotide sequence of 92.1 gene of Mccp genome.Visual dyes of Hydroxynaphthol blue(HNB) or fluorescent dyes(Eva green) was added into the reaction system as indictors respectively,a visual LAMP(V-LAMP) and a realtime fluorescence LAMP(RF-LAMP) were established.The sensitivity,specificity and reproducibility as well as the detection effectiveness in goat samples of these two LAMP assays were evaluated.The results indicated that the two methods are both capable of detecting 1.0×10 copies/μL of standard plasmid,and they all have no cross-reactivity with other mycoplasmas and bacteria,such as M.mycoides subsp.capricolum,M.ovipenumoniae,Pasteurellamultocida,Klebsiella Pneumoniae etc..The coefficient of variation of both assays is both less than 1%.A total of 36 goat tissue samples from experimental infection and clinic cases were tested by the two methods,both detected 3 positives indicating a positive rate of 9.09%,which was consistent with the result from a Taq Man RT-PCR published.In conclusion,the V-LAMP and RF-LAMP established in this experiment can be used for rapid detection of Mccp.It provided new methods for the epidemiology investigation and diagnosis of Mccp infection.