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目的基于环介导等温核酸扩增法(LAMP)建立副溶血性弧菌tlh基因的快速检测方法。方法以副溶血性弧菌tlh基因为扩增的靶序列分别设计外引物、内引物和环引物各1对,通过引物特异性识别tlh基因上的6个独立区域在恒温条件下扩增靶序列,快速检测副溶血性弧菌。并对该法进行灵敏度、特异度试验,将该方法与荧光定量PCR法检出率进行统计学比较。结果 LAMP法最低检出限为10 fg/μl,灵敏度是荧光定量PCR的10倍;特异度实验中除检出2株副溶血性弧菌外,6株非副溶血性弧菌均未检出;LAMP法与荧光定量PCR法检出率差异无统计学意义(P>0.05)。结论 LAMP法具有高特异性、高效性、快速简便易检测等特点,适合现场快速检测,在食物中毒暴发处置中有深远的经济价值。
Objective To establish a rapid method for the detection of tlh gene of Vibrio parahaemolyticus based on the ring-mediated isothermal amplification of nucleic acid (LAMP). Methods The outer primers were designed by using the target sequence amplified by the Vibrio parahaemolyticus tlh gene. The inner primers and the loop primers were designed as 1 pair respectively. The primers were used to specifically target 6 independent regions on the tlh gene to amplify the target sequence , Rapid detection of Vibrio parahaemolyticus. The sensitivity and specificity of the method were tested, and the detection rate of this method was compared with that of fluorescence quantitative PCR. Results The minimum detectable limit of LAMP assay was 10 fg / μl, and the sensitivity was 10 times of that of real-time PCR. Six non-hemolytic vibrio strains were not detected in the specificity test except two Vibrio parahaemolyticus There was no significant difference between LAMP method and real-time PCR (P> 0.05). Conclusion The LAMP method has the characteristics of high specificity, high efficiency, quick and easy detection and so on. It is suitable for rapid detection in the field and has far-reaching economic value in food poisoning and outbreak treatment.