Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimp

• Published in: Applied microbiology and biotechnology 2015-08, Vol.99 (15), p.6451-6462
• Main Authors: Zhang, Zhaohuan, Liu, Haiquan, Lou, Yang, Xiao, Lili, Liao, Chao, Malakar, Pradeep K, Pan, Yingjie, Zhao, Yong
• Format: Article
• Language: English
• Subjects: Analysis; Bacteria; Biomedical and Life Sciences; Biotechnology; Cold Temperature; Contamination; Decapoda; Food; Food Microbiology - methods; Food safety; Food Storage; Life Sciences; Listeria; Listeria monocytogenes; Listeria monocytogenes - isolation & purification; Listeria monocytogenes - physiology; Methods; Methods and Protocols; Microbial Genetics and Genomics; Microbial Viability; Microbiology; monitoring; Multiplex Polymerase Chain Reaction - methods; Pathogens; PCR; propidium; Public health; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; Research parks; Safety and security measures; Seafood; Seafood - microbiology; seafoods; Sensitivity and Specificity; Shellfish; shrimp; Studies; Temperature; Time Factors; Vibrio parahaemolyticus; Vibrio parahaemolyticus - isolation & purification; Vibrio parahaemolyticus - physiology
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-015-6715-x

A novel TaqMan-based multiplex real-time PCR method combined with propidium monoazide (PMA) treatment was firstly developed for the simultaneous quantification of viable Vibrio parahaemolyticus and Listeria monocytogenes in raw shrimp. The optimization of PMA concentration showed that 100 μM was considered optimal to effectively inhibit 10⁸ CFU/mL dead cells of both bacteria. The high specificity of this method was confirmed on tests using 96 target and non-target strains. The optimized assay could detect as low as 10¹–10² CFU/g of each strain on the artificially contaminated shrimp, and its amplification efficiencies were up to 100 and 106 % for V. parahaemolyticus and L. monocytogenes, respectively. Furthermore, this assay has been successfully applied to describe the behavior of these two pathogens in raw shrimps stored at 4 °C. In conclusion, this PMA TaqMan-based multiplex real-time PCR technique, where the whole procedure takes less than 5 h, provides an effective and rapid tool for monitoring contamination of viable V. parahaemolyticus and L. monocytogenes in seafood, improving seafood safety and protecting public health.