Detection of Vibrio parahaemolyticus in tropical shellfish by SYBR green real-time PCR and evaluation of three enrichment media.

摘要

A rapid, sensitive and highly reproducible SYBR green based real-time PCR assay was developed for detection of tdh positive pathogenic Vibrio parahaemolyticus. Minimum detection limit was 0.1 pg of pure V. parahaemolyticus genomic DNA with typical R2 values >0.99 and coefficient of variation (CV) values ranging from 1.2 to 4.2 on three different days. The method was also used to evaluate the effect of three different enrichment media alkaline peptone water (APW), sodium taurocholate (ST) broth and salt polymyxin broth (SPB) on detection of V. parahaemolyticus. Crude lysates were directly used for real-time PCR. Without enrichment, the detection limit of pure cultures was 101 CFU/ml for ST broth and 102 CFU/ml in APW and SPB but for shrimp homogenates spiked with pure culture, the minimum detection limit was 102 CFU/ml for all three broths with a linear detection range of 102-106. Without enrichment, detection in ST broth was more efficient than APW and SPB. After 6 h enrichment, limit of detection was found to be 1 CFU in all three media. However, for iced shrimp, the limit of detection was 102 after 6 h enrichment. No significant difference was seen between different enrichment media with respect to tdh gene detection of V. parahaemolyticus. The methodology developed here can be useful for rapid detection of tdh positive V. parahaemolyticus by laboratories involved in monitoring programmes for pathogenic V. parahaemolyticus. All rights reserved, Elsevier.