猪附红细胞体eno基因可视化LAMP检测方法的建立与应用

Establishment and application of LAMP detection method for eno gene of Mycoplasma suis

为建立一种快速、便捷、可基层现场检测猪附红细胞体(M.suis)的环介导等温扩增方法(LAMP),本研究参考GenBank已登录的M.suis基因组(NC_015153),以其α-烯醇化酶(eno)基因为目的基因设计合成4条特异引物,经反应条件优化,初步建立了检测M.suis的LAMP方法,其内外引物比为1􀏑6,可在63℃,反应50 min时通过SYBR Green I染料直接经肉眼判定结果。利用该方法检测弓形虫、大肠杆菌、猪链球菌I型、牛源瑟氏泰勒虫和M.suis,结果显示均无交叉反应,特异性强;将新提取的重组质粒pMD-eno 10倍倍比稀释(100~10-7),利用本研究建立的LAMP方法进行敏感性试验,结果显示,该方法对重组质粒pMD-eno最低检测限为30拷贝/μL,比普通PCR检测方法敏感度高100倍,敏感性高;利用建立的LAMP方法对同一时间和不同时间提取的M.suis基因组(101~106)进行组内组间重复试验,结果显示组内与组间检测结果均为阳性,重复性好。利用该方法对84份疑似感染M.suis的猪血液样品进行检测,结果显示LAMP检测结果与PCR方法检测结果一致,表明本研究建立的LAMP检测方法可应用于M.suis临床样品检测。本研究建立的LAMP方法操作简单、特异、灵敏和重复性好,经肉眼可判读结果,适合猪场M.suis感染的现场检测。

In order to establish a fast,convenient loop-mediated isothermal amplification method(LAMP)that can detect Mycoplasma suis(M.suis)at the grassroots level,this study referred to the M.suis genome(NC_015153)registered in GenBank.Theα-enolase(eno)gene is the target gene and 4 specific primers were designed and synthesized based on this gene.After optimization of the reaction conditions,the LAMP method established in this study has a ratio of inner and outer primers of 1:6,which can using SYBR Green Ⅰ at 63℃for 50 minutes.The result is directly judged by naked eyes.This method has no crossreactivity with Toxoplasma gondii,Escherichia coli,Streptococcus suis type I,Theileria sergiomas,and Eperythrozoites of bovine origin,and has strong specificity.The sensitivity test showed that the minimum detection concentration was 30 copies/μL on M.suis eno.The detection limit was 30 copies/μL,which was 100 times more sensitive than the ordinary PCR detection method.The result of variation within and between groups was consistent,indicating that its repeatability was good.The established LAMP detection method was used for 84 porcine blood samples suspected of being infected with M.suis,and the results showed that the LAMP test results were consistent with the PCR method test results,indicating that the LAMP test established in this study could be applied to the detection of M.suis clinical samples.The LAMP method established in this study was simple,specific,sensitive and repeatable,and the results could be read by the naked eye.It was suitable for field detection and field application of M.suis infection in pig farms.