blaCARB-17-based LAMP assay for detectingVibrio parahaemolyticus in aquatic products
HU Yuanqing1,2*, SHEN Zichen1, LI Fengxia1, LYU Linxue1, ZHOU Zanhu3
1(School of Biological Science and Biotechnology,Minnan Normal University,Zhangzhou 363000,China); 2(Jiangsu Key Laboratory of Zoonosis,Yangzhou University,Yangzhou 225000,China); 3(Comprehensive Technical Service Center,Zhangzhou Customs,Zhangzhou 363000,China)
Abstract: A highly specific loop-mediated isothermal amplification (LAMP) assay targeting theblaCARB-17 gene was developed for rapid and sensitive detection ofVibrio parahaemolyticus in aquatic products.The assay was optimized and conducted at 63 ℃ for 60 min usingBacillus stearothermophilus(Bst) DNA polymerase.Amplification was analyzed via 20 g/L agarose gel electrophoresis followed by SYBR Green Ⅰ staining.The specificity was determined by detectingV.parahaemolyticus ATCC 17802 and other 5 foodborne pathogens.The sensitivity was evaluated by detecting diluted genome DNA samples.The reliability was proved in both simulation experiment using experimentally contaminated shrimp samples and detection of aquatic samples from fish markets.The optimum detection condition was:2.4 mmol/L Mg2+,0.96 mmol/L dNTPs,4.8 UBst DNA polymerase,the ratio of inner and outer primer was 8:1,and react at 65 ℃ for 60 min.The result of specificity showed thatV.parahaemolyticus ATCC 17802 was positive,and other 5 control strains were negative.The detection limit of LAMP assay was 3.64×102 ng/μL,and the detection limit in the simulation experiment was 10 CFU/mL.The LAMP assay showed 100% consistency with conventional PCR for detecting practical samples.The LAMP assay established in this experiment is convenient,sensitive and specific,and is suitable for rapid in-field detection ofV.parahaemolyticus.
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