羊丝状支原体簇环介导等温扩增检测方法的建立

Detection of Mycoplasma mycoides Cluster by Loop-mediated Isothermal Amplification

本试验利用环介导等温扩增技术(LAMP)建立了羊丝状支原体簇的快速检测方法,该方法以羊丝状支原体簇成员的保守性基因16S rRNA为靶序列,设计了4条特异性引物,在65℃等温条件下,60 min一步完成反应。在反应管中预先添加羟基萘酚蓝(HNB),阳性呈蓝色,阴性呈紫色。丝状支原体簇成员——丝状支原体山羊亚种(Mmc)、丝状支原体丝状亚种LC型(Mmm LC)、山羊支原体山羊肺炎亚种(Mccp)的LAMP检测为阳性;对其他病原菌没有交叉反应。以Mmc的核酸为模板进行灵敏度检测,LAMP的最低检出限为10 pg/μL。结果表明,本试验建立了一种特异、敏感、快速、简便的羊丝状支原体簇的LAMP方法。

In this study,a sensitive loop-mediated isothermal amplification（LAMP） was developed for rapidly detecting Mycoplasma mycoides cluster.A set of four primers,two outer and two inner primers,were designed from genomic sequences according to the conserved region of the 16S rRNA gene of Mycoplasma mycoides cluster.The reaction was performed in one step in a single tube at 65 ℃ for 60 min,with hydroxynaphthol blue（HNB） dye added prior to amplification.Blue-positive results were significantly different from the purple-negative.The assay could amplify from Mycoplasma mycoides cluster members,Mmc,Mmm LC and Mccp,but no cross-reaction from other bacteria.The sensitivity test showed that it could detect 10 pg/μL DNA from Mmc strain PG3.The study had developed a diagnostic procedure which was rapid and sensitive for Mycoplasma mycoides cluster.