2型猪链球菌89K毒力岛Ⅳ型分泌系统LAMP检测方法的建立

目的建立针对高致病性2型猪链球菌(Streptococcus suis 2,SS2)89K毒力岛Ⅳ型分泌系统的环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法。方法根据国内流行株特有89K毒力岛编码的Ⅳ型分泌系统(T4SS-89K)的virB4-89K基因保守区域设计并合成LAMP引物,通过优化反应体系和扩增条件建立T4SS-89K快速检测方法,对方法的特异性、敏感性进行评估。结果优化的LAMP反应体系具有良好的扩增效率,检测灵敏度为1.53×101拷贝/反应,全部扩增检测可在60min内完成;建立的LAMP法具有良好的特异性,与不含T4SS-89K的猪链球菌(包括1/2型、1型、3~33型),以及其他常见9种对照菌均不发生特异性扩增,而与1998和2005年疫情现场分离的高致病性SS2均可发生特异性扩增。结论建立的LAMP方法具有特异、灵敏,设备要求简单等特点,可用于高致病性SS2快速检测。

Use of loop-mediated isothermal amplification to detect highly virulent Streptococcus suis serotype 2

Objective This study sought to develop a system of loop-mediated isothermal amplification （LAMP） for rapid and specific detection of highly virulent Streptococcus suis serotype 2. Methods Two sets of specific primers were designed and synthesized in accordance with the target gene, the virulence gene virB4-89K in the type Ⅳ secretion system （T4SS） gene of 89K PAl. The more effective set was selected and reaction conditions were optimized. Evaluation included testing samples of infected blood. Specificity was evaluated using 33 strains of S. suis serotypes 1/2, 1, and 8 to 33, a- long with 9 strains of other bacterial species related to S. suis 2. The detection limit for LAMP was assessed by serial di- lution using the same target gene. Results The reaction system had good amplification efficiency and was completed within 60 minutes. Sensitivity for the gene virB4-89K in T4SS-89K was 1.53 × 10-1 copies/reaction. No corresponding am- plification was noted when using 42 strains of different bacterial species as a template, indicating that LAMP was highly specific. Blood from individuals infected with S. suis 2 tested positive. Conclusion This LAMP method has high speci- ficity and sensitivity and requires only simple laboratory equipment. It is suitable for the rapid detection of highly virulent S. suis serotype 2.