Absolute quantification of Vibrio parahaemolyticus by multiplex droplet digital PCR for simultaneous detection of tlh, tdh and ureR based on single intact cell. (August 2020)

• Record Type: Journal Article
• Authors: Lei, Shuwen; Gu, Xiaokui; Zhong, Qingping; Duan, Linjie; Zhou, Aimei
• Is Part Of: Food control. Volume 114(2020)
• Publication Date: 2020-08
• Subjects: Vibrio parahaemolyticus -- multiplex ddPCR -- Quantitative PCR -- Single intact cell -- Absolute quantification -- Rapid detection; Food -- Quality -- Periodicals; Food -- Analysis -- Periodicals; Food handling -- Periodicals; Food industry and trade -- Quality control -- Periodicals; Aliments -- Industrie et commerce -- Qualité -- Contrôle -- Périodiques; Aliments -- Qualité -- Périodiques; Aliments -- Analyse -- Périodiques; Hygiène alimentaire -- Périodiques; Food -- Analysis; Food handling; Food -- Quality; Periodicals; Electronic journals; 664.07
• DOI: 10.1016/j.foodcont.2020.107207
• Languages: English
• ISSNs: 0956-7135

Abstract

Abstract: Vibrio parahaemolyticus, a marine food-borne pathogen, has been proved to be a significant cause of human gastrointestinal disorders worldwide. In this study, a method of multiplex droplet digital PCR (ddPCR) based on primer and probe sequences of the tlh, tdh and ureR genes were developed and evaluated for the reliable quantification of V. parahaemolyticus cells in seafoods. The specificities of all primers and probes used in this study were validated on three standard strains of V. parahaemolyticus, 10 strains of Vibrio spp., and 22 strains of other bacteria by ddPCR and quantitative PCR (qPCR). Then the ddPCR system, primers, probe concentration and amplification procedures were optimized, and the templates with cell and genomic DNA (gDNA) were compared. The results showed that using cell as template could benefit for multiplex ddPCR, which performed higher linkage among three genes. This method improved sensitivity, specificity, accuracy, convenience, and reproducibility for the detection of V. parahaemolyticus, and the limit of detection (LOD) was 15 CFU/mL. In addition, the applicability of this method was compared with plate count and qPCR, and then verified to detect artificially contaminated seafood samples containing different concentrations of V. parahaemolyticus . The results indicated that the established method is stable, accurate, sensitive, range-wide, and has the potential to detect the three different genes of V. parahaemolyticus in food samples. Highlights: Multiplex ddPCR was developed and evaluated to directly quantify V. parahaemolyticus. The assay exhibited significant specificity, sensitivity and accuracy. The method was used to monitor three genes and distinguish different V. parahaemolyticus. The multiplex ddPCR based on single intact cell is better than that of gDNA. The applicability of the assay was verified to detect spiked seafood samples.