应用LAMP技术鉴定含89K毒力岛2型猪链球菌菌株

Discriminating strains of Streptococcus suis serotype 2 of 89K pathogenicity island by Loop-Mediated Isothermal Amplification technology

目的应用LAMP(Loop-Mediated Isothermal Amplification)技术鉴别含89K毒力岛2型猪链球菌(Streptococ-cus suisserotype2,S.suis2)菌株。方法根据S.suis2中国分离株(05ZYH33)89K毒力岛中基因序列设计4条引物(2条内引物、2条外引物);应用LAMP,对21株S.suis2、18株其它链球菌、9株葡萄球菌、5株其它菌株及49份未知样本进行检测,并对LAMP的最低检测限进行测试,结果通过肉眼目测或电泳检测,并将结果与PCR/定量PCR结果比较。结果该方法仅对含89K毒力岛的中国S.suis2特有株检测为阳性,而其它菌株经LAMP检测均为阴性,试验表明本方法特异性好,其最低检测限为24CFU/反应,无需特殊设备,结果通过肉眼即可判断,从菌株核酸的提取至检测完成仅需2h左右。应用本方法对49份不同来源样本进行实际考核,其中32份不明发热病人呼吸道咽拭子应急样本及15份正常猪扁桃体咽拭子样本经检测89K毒力岛基因均为阴性,2份疑似SS2病人血液应急样本中一份检测到该毒力岛基因,该结果与普通PCR/定量PCR检测结果一致。结论成功建立了检测S.suis289K毒力岛的LAMP方法 ,该方法特异性好,操作简便,无需特殊的仪器设备,操作简单方便,极适合在我国广大基层实验室(包括基层医院/动物检疫等部门)开展应用。

Streptococcus suis serotype 2 Chinese strains with 89K pathogenicity island gene was identified by loop-mediated isothermal amplification（LAMP）method in this study.Four primers（two inside and two outside）according to the DNA sequence of 89K pathogenicity island gene from Streptococcus suis serotype 2 strain（05ZYH33）were designed,and LAMP reaction system were improved.A total of 21 strains of Streptococcus suis serotype 2,18 strains of other Streptococcus,9 strains of Staphylococcus,5 strains of other bacterium and 49 unknown samples were used to assess the specificity of LAMP method,and the minimum detection limit were tested.Results were observed by naked eye or electrophoresis,and compared with detection results from PCR/Quantitative PCR.Detection results showed that only Chinese strains of Streptococcus suis serotype 2 containing 89K pathogenicity island gene were positive,while other bacterium strains were negative.LAMP method revealed good specificity and the minimum detection limit was 24CFU/reaction.The result could be judged by naked eyes without any special instrument and it just took about 2 hours to finish the test from the DNA extract.Then this method was assessed by detecting 49 samples of different sources.Among 32 emergency samples of throat swab from unknown fever patients,2 emergency samples from suspected Streptococcus suis serotype 2 patients＇ blood and 15 samples of throat swab from natural pigs＇ amygdalae,only 1 positive was detected from the 2 emergency samples from suspected Streptococcus suis serotype 2 patients＇ blood.The result of LAMP was same as that of common PCR/Quantitative PCR.It＇s indicated that this successful LAMP method could rapidly and specifically detect and identify the gene segment containing 89K pathogenicity island of Streptococcus suis serotype 2 Chinese strain without any special instrument.As the operation procedure is simple and convenient,it is very proper for application in laboratories in basic hospitals and animal quarantine stations at basic level in China.