Development of colorimetric multiplex loop mediated isothermal amplification combined with lateral flow dipstick (mLAMP-LFD) assay for the identification of TDH toxin-producing Vibrio parahaemolyticus

IF 4 2区农林科学 Q2 CHEMISTRY, APPLIED

Journal of Food Composition and Analysis Pub Date : 2024-11-01 DOI:10.1016/j.jfca.2024.106924

Soottawat Benjakul , Phutthipong Sukkapat , Suriya Palamae , Prashant Singh , Mingkwan Yingkajorn , Jirayu Buatong , Jirakrit Saetang

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引用次数: 0

Abstract

Vibrio parahaemolyticus in seafood is associated with gastroenteritis diseases worldwide. Rapid and sensitive detection methods of the pathogenic strain are essential for monitoring and preventing the diseases. Colorimetric multiplex loop-mediated isothermal amplification combined with lateral flow dipstick assay (mLAMP-LFD) was developed to differentiate pathogenic strains from non-pathogenic V. parahaeholyticus. Thermolabile hemolysin (TLH) and thermo-stable direct hemolysin (TDH) genes were designed and used for mLAMP-LFD assay. The assay specificity was validated using strains belonging to 11 species of other foodborne pathogens, while the sensitivity was evaluated by using a DNA template ranging from 0.005 to 0.4 ng/reaction. Shrimp samples inoculated at 10¹, 10², and 10³ CFU/ 10 g, and enrichment times of 0, 2, 4, 6, and 8 h were used for assay validation. The standardized assay accurately identified 20 V. parahaemolyticus strains and showed no cross-amplification. The assay’s limit of detection was 0.02 ng and took 30 min to complete. Shrimp samples inoculated at 10 CFU of V. parahaemolyticus could be detected following a four-hour enrichment. In conclusion, the high sensitivity and specificity of the mLAMP-LFD assay developed in the present study could be used to identify pathogenic strains of V. parahaemolyticus.

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开发用于鉴定产 TDH 毒素副溶血性弧菌的比色多反应环介导等温扩增结合侧流浸量尺（mLAMP-LFD）测定法

海产品中的副溶血性弧菌与世界各地的肠胃炎疾病有关。快速灵敏地检测致病菌株对监测和预防疾病至关重要。为了区分致病性菌株和非致病性副溶血性弧菌，研究人员开发了比色多重环介导等温扩增结合侧向流动点滴检测法（mLAMP-LFD）。设计了热吸收溶血素（TLH）和热稳定直接溶血素（TDH）基因，并将其用于 mLAMP-LFD 检测。使用属于 11 种其他食源性病原体的菌株验证了该检测方法的特异性，同时使用 0.005 至 0.4 纳克/反应的 DNA 模板评估了该检测方法的灵敏度。虾样品的接种量分别为 101、102 和 103 CFU/ 10 克，富集时间分别为 0、2、4、6 和 8 小时。标准化检测方法准确鉴定了 20 种副溶血性弧菌菌株，未发现交叉扩增现象。该检测方法的检测限为 0.02 毫微克，需 30 分钟完成。对虾样品接种 10 CFU 副溶血弧菌后，经过四小时富集，即可检测到副溶血弧菌。总之，本研究开发的 mLAMP-LFD 检测方法灵敏度高、特异性强，可用于鉴定副溶血性弧菌的致病菌株。

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来源期刊

Journal of Food Composition and Analysis 工程技术-食品科技

CiteScore

6.20

自引率

11.60%

发文量

601

审稿时长

53 days

期刊介绍： The Journal of Food Composition and Analysis publishes manuscripts on scientific aspects of data on the chemical composition of human foods, with particular emphasis on actual data on composition of foods; analytical methods; studies on the manipulation, storage, distribution and use of food composition data; and studies on the statistics, use and distribution of such data and data systems. The Journal''s basis is nutrient composition, with increasing emphasis on bioactive non-nutrient and anti-nutrient components. Papers must provide sufficient description of the food samples, analytical methods, quality control procedures and statistical treatments of the data to permit the end users of the food composition data to evaluate the appropriateness of such data in their projects. The Journal does not publish papers on: microbiological compounds; sensory quality; aromatics/volatiles in food and wine; essential oils; organoleptic characteristics of food; physical properties; or clinical papers and pharmacology-related papers.