环介导等温扩增技术快速检测副溶血性弧菌

目的建立一种检测副溶血性弧菌（Vp）快速且特异的环介导等温扩增（LAMP）检测方法。方法针对副溶血性弧菌不耐热溶血素（tlh）基因特异性序列的6个位点设计4条LAMP引物，65℃保温约60min，完成对副溶血性弧菌的扩增，扩增产物经肉眼、SYBR Green Ⅰ染色、电泳和酶切鉴定。利用LAMP和普通PCR方法同时检测1株副溶血性弧菌和12株非副溶血性弧菌来验证LAMP方法的特异性；将副溶血性弧菌菌液作一系列10倍稀释后用LAMP和PCR方法进行检测，比较两者敏感性。结果1株副溶血性弧菌出现LAMP扩增反应：通过肉眼、SYBR Green Ⅰ染色和电泳均能观察到LAMP扩增产物的出现，酶切证实了LAMP产物的特异性；12株非副溶血性弧菌未出现LAMP扩增。LAMP检测tlh基因的特异性和敏感性与普通PCR相同，LAMP检测tlh基因的检测下限为15cfu／mL。结论建立了一种快速、敏感、特异的副溶血性弧菌检测方法，适合日常监测及快速检测的需要。

Rapid detection of vibrio parahaemolyticus with loop-mediated isothermal amplification

Objective To develop a loop-mediated isothermal amplification(LAMP) assay for the rapid and specific detection of vibrio parahaemolyticus(Vp). Methods Four primers which recog-nized 6 distinct regions on the thermolabite hemolysin(tlh) gene of Vp were designed and used for LAMP assay. Vp DNA was amplified under isothermal conditions(65 ℃) for 60 rain, then the ampli-fied product was judged by naked eye, SYBR Green I staining, electrophoretie analysis and restriction digestion. To evaluate the specificity of the assay, 1 strain of Vp and 12 strains of non-Vp were tested by LAMP and conventional PCR simultaneously. In addition, the detection limit of LAMP was com-pared with that of PCR by using the Vp strain, that were 10-fold serially diluted and was amplified by LAMP and PCR. Results With 1 strain of Vp, the naked eye, SYBR Green I staining and electro-phoretic analysis could detect the products in the LAMP assay. The specificity of LAMP products was confirmed by digestion of LAMP products using restriction enzymes. Amplification of LAMP was not observed when 12 strains of non-Vp were tested. The specificity and sensitivity of LAMP were similar to those of conventional PCR assay and the detection limit of LAMP assay was 15 cfu/mL. Conclusion These results indicate that the LAMP assay is a rapid, specific and sensitive detection method for Vp. The method is suitable fo: daily monitoring and rapid diagnosis of Vp.