副溶血性弧菌
环介导等温扩增
量油尺
不耐热的
放大器
生物
微生物学
检出限
弧菌
色谱法
聚合酶链反应
分子生物学
细菌
化学
基因
DNA
遗传学
尿
生物化学
酶
作者
Piyanuch Prompamorn,Paisarn Sithigorngul,Sombat Rukpratanporn,Siwaporn Longyant,Pattarin Sridulyakul,Parin Chaivisuthangkura
标识
DOI:10.1111/j.1472-765x.2011.03007.x
摘要
The current study was aimed to develop a loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay for rapid and specific detection of Vibrio parahaemolyticus.Biotinylated LAMP amplicons were produced by a set of four designed primers that recognized specifically the V. parahaemolyticus thermolabile haemolysin (tlh) gene followed by hybridization with an FITC-labelled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65 °C. The LAMP-LFD method accurately identified 28 isolates of V. parahaemolyticus but did not detect 24 non-parahaemolyticus Vibrio isolates and 35 non-Vibrio bacterial isolates. The sensitivity of LAMP-LFD for V. parahaemolyticus detection in pure cultures was 120 CFU ml⁻¹. In the case of spiked shrimp samples without enrichment, the detection limit for V. parahaemolyticus was 1·8 x 10³ CFU g⁻¹ or equivalent to 3 CFU per reaction while that of conventional PCR was 30 CFU per reaction.The established LAMP-LFD assay targeting tlh gene was specific, rapid and sensitive for identification of V. parahaemolyticus.The developed LAMP-LFD assay provided a valuable tool for detection of V. parahaemolyticus and can be used effectively for identification of V. parahaemolyticus in contaminated food sample.
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