霍乱弧菌毒力基因LAMP方法快速检测

姚文山; 吴家林; 沙丹; 马广源; 张敬平

doi:10.11847/zgggws2015-31-01-20

霍乱弧菌毒力基因LAMP方法快速检测

Rapid detection of Vibrio cholerae toxic genes with loop-mediated isothermal amplification method

摘要

摘要: 目的建立快速、特异检测霍乱弧菌的环介导等温扩增(LAMP)方法。方法针对霍乱弧菌毒素基因(ctxA)设计4条引物,建立环介导等温扩增检测方法,优化反应体系,测定方法的特异性和灵敏度,并进行鱼肉模拟样品和实际样品检测。结果 LAMP最佳反应条件为8.0 mmol/L Mg²⁺、0.6 mmol/L dNTPs、1.6 μmol/L FIP和BIP(2条内引物)、0.2 μmol/L F3和B3(2条外引物);对细菌纯培养物的检测灵敏度为10 cfu/mL,对鱼肉模拟样品检测灵敏度为10² cfu/g;在16份实际样品中检测出4份ctxA基因阳性。结论建立的LAMP方法灵敏度高、特异性强,可快速地检测食品样品中霍乱弧菌。

Abstract: Objective To establish a loop-mediated isothermal amplification(LAMP) method for rapid detection of Vibrio cholerae.Methods Four primers were designed specifically according to ctxA gene of Vibrio cholerae.The LAMP system was developed and optimized.The specificity and sensitivity of this system were evaluated.Artificially contaminated fish and practical samples were detected.Results The LAMP method was highly sensitive and specific.The sensitivity of the LAMP detection was 10 cfu/ml of bacteria samples and 10² cfu/g of Vibrio cholerae in artificially contaminated fish.The assay identified 4 Vibrio cholerae,with ctxA primers from 16 practical samples.Conclusion A rapid,specific and sensitive LAMP method for detection of Vibrio cholerae was established and could be used in rapid detection of Vibrio cholerae in food samples.

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