快速检测猪链球菌的环介导等温扩增方法

目的建立猪链球菌(S. suis)的环介导等温扩增方法(LAMP)。方法通过BLASTn序列比对确定较为保守的S. suis种特异基因,用软件PrimerExplorer V4设计针对靶基因的LAMP试验引物;分别用煮沸法和试剂盒提取法提取基因组做敏感性试验,并用62株非S. suis菌株评价了实验的特异性;再用100株分离自不同地区不同时间的S. suis菌株评价该方法的可靠性。结果确定了S. suis种特异性基因dnaN为靶基因,并针对该基因建立了检测S. suis的LAMP方法。敏感性实验中,每个反应的检测下限是31.25 fg纯DNA,相当于14个拷贝;煮沸法的检测下限是每个反应27 CFU。在对62株非S. suis菌株与有争议的S. suis菌株的特异性评价实验中,未发现有假阳性。结论成功建立了针对dnaN基因检测S. suis的LAMP方法。

Loop-mediated isothermal amplification assay targeting dnaN gene for rapid detection of Streptococcus suis

Streptococcus suis (S. suis) is an important emerging zoonotic agent with high public health significance. S. suis is routinely identified using culture and biochemical assays and a PCR test targeting gdh coding for glutamate dehydrogenase. Here we found the dnaN gene to be more conserved and specific than the gdh gene in S. suis. Thus, the dnaN gene was chosen as the target gene to design loop-mediated isothermal amplification (LAMP) assays for the rapid, specific, and sensitive detection of S. suis. The LAMP procedure used 63 ℃ for 60 min. No false-positives were observed for the 62 non- S. suis strains and the four controversial reference strains used to evaluate specificity of the assay. The limit of detection for pure S. suis genomic DNA was approximately 31.25 fg, equal to 14 copies per reaction; the limit of detection using the boiling method was 27 colony forming units (CFU) per reaction. All of the 100 S. suis strains used in the evaluation assay were positive in the LAMP assay. This assay has potential for use in field conditions for epidemic prevention and entry-exit inspection.