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A Novel Reverse-Transcriptase Real-Time PCR Method for Quantification of Viable Vibrio Parahemolyticus in Raw Shrimp Based on a Rapid Construction of Standard Curve Method

Mengtong Jin, Haiquan Liu, Wenshuo Sun, Qin Li, Zhaohuan Zhang, Jibing Li,Yingjie Pan, Yong Zhao

Vibrio parahemolyticus is an important pathogen that leads to food illness associated seafood. Therefore, rapid and reliable methods to detect and quantify the total viable V. parahaemolyticus in seafood are needed. In this assay, a RNA-based real-time reverse-transcriptase PCR (RT-qPCR) without an enrichment step has been developed for detection and quantification of the total viable V. parahaemolyticus in shrimp. RNA standards with the target segments were synthesized in vitro with T7 RNA polymerase and subsequently converted to cDNA for establishing the standard curve to quantify V. parahaemolyticus. The reaction efficiency was 94.56%, and was in the acceptable range. Without pre-enrichment, the sensitivity of RT-qPCR on quantifying V. parahaemolyticus in shrimp was 58 cfu/g, approximately. In addition, the survival of V. parahaemolyticus in spiked shrimp samples was quantified by RT-qPCR, DNA-based qPCR and standard plate count method. The quantification results of RT-qPCR have shown a good statistical correlation (R2=0.96) when compared to the standard plate count method. However, the correlation coefficient between plate count method and DNA-based qPCR was only 0.85 for quantifying V. parahaemolyticus. Based on the results, this new method could be an effective way for providing reliable quantitative data on viable V. parahaemolyticus in shrimp.