霍乱弧菌CT毒素环介导等温扩增技术快速检测方法的建立

Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae by using loop-mediated isothermal amplification

目的将一种新颖的核酸扩增技术——环介导等温扩增技术(LAMP)应用于霍乱弧菌毒素基因ctxA的快速检测。方法针对霍乱弧菌毒素基因ctxA设计6条特异性引物(两条内引物、两条外引物和两条环引物)进行LAMP扩增,对扩增反应进行优化。并考核方法的敏感性和特异性。结果最佳反应时间为60 min,反应温度为65℃。对8种相关肠道细菌进行LAMP扩增,仅含毒素基因ctxA的霍乱弧菌得到阳性扩增结果,证明引物具有很高的特异性。霍乱弧菌基因组DNA和纯培养物的检测灵敏度分别约为68 fg和42 cfu/ml。对模拟样品进行直接检测,检测限为72 cfu/g。结论该方法检测霍乱弧菌毒素基因ctxA特异性强、灵敏度高,并且操作简便、检测成本低,1 h即可完成,适合基层检验部门使用。

Objective To establish a loop-mediated isothermal mplification（LAMP） assay for the rapid and specific detection of cholera toxin-producing Vibrio cholerae.Methods A set of 6 primers,including 2 outer primers,2 inner primers and 2 loop primers,were designed specifically to recognize the ctxA gene of V.cholerae.The reaction conditions were optimize.Specificity and sensitivity of LAMP were tested by using cholera toxin-producing V.cholerae,non-cholerae vibrio isolates and non-vibrio bacterial isolates.Results The optimized time and temperature conditions for the LAMP assay were 60 min and at 65 ℃,respectively.The LAMP could accurately identify the V.Cholerae isolates carrying the ctxA gene,but fail to detect other non-cholerae vibrio isolates and non-vibrio bacterial isolates.The detection limits of the method were 68 fg of purified genomic DNA/reaction and 42 cfu/ml.In addition,this method was applied to detect artificially contaminated samples and the detection limit was 72 cfu/g for the artificially contaminated samples without precultivation.Conclusion The results suggested that the LAMP is sensitive,specific,inexpensive and rapid in the detection of V.cholerae carrying ctxA gene,which can used in basic clinical laboratory.