The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus
The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus
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DOI：
10.1111/j.1472-765x.2011.03007.x
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发表时间：
2011-04-01
影响因子：
2.4
通讯作者：
Chaivisuthangkura, P.
中科院分区：
文献类型：
--
作者：
Prompamorn, P.;Sithigorngul, P.;Chaivisuthangkura, P.
目的：建立环介导等温扩增(LAMP)结合侧向流动试纸(LFD)检测副溶血性弧菌的方法。方法与结果：设计了4条能特异性识别副溶血弧菌tlh基因的引物，通过与FITC标记的探针杂交和LFD检测，获得生物素化的LAMP扩增产物。LAMP-LFD方法能准确鉴定出28株副溶血性弧菌，但未检出24株非副溶血性弧菌和35株非弧菌。LAMP-LFD检测纯培养副溶血性弧菌的灵敏度为120cfu ml-1。结论：针对tlh基因建立的LAMP-LFD方法对副溶血性弧菌的检测具有特异性、快速、灵敏等特点，为副溶血性弧菌的检测提供了一种有价值的检测方法，可有效用于食品样品中副溶血性弧菌的检测。
Aims: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.Methods and Results: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 degrees 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 center dot 8 x 103 CFU g-1 or equivalent to 3 CFU per reaction while that of conventional PCR was 30 CFU per reaction.Conclusions:The established LAMP-LFD assay targeting tlh gene was specific, rapid and sensitive for identification of V. parahaemolyticus.Significance and Impact of the Study: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.