Food and Fermentation Industries >

2022 , Vol. 48 >Issue 1: 247 - 252

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.027703

Development and optimization of PMA-qPCR for the quantitative detection of viable but non-culturable Vibrio parahaemolyticus

  • PENG Linyuan ,
  • LIN Hong ,
  • WANG Jingxue
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  • (College of Food Science and Engineering,Ocean University of China,Qingdao 266000,China)

Received date: 2021-04-16

  Revised date: 2021-05-12

  Online published: 2022-01-27

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Abstract

Vibrio parahaemolyticus might turn to a viable but non-culturable (VBNC) state under some natural and food processing conditions where bacteria can hardly grow, which cannot be detected by conventional culture-based methods. Therefore, the development and optimization of the VBNC bacteria detection technology is significant for the prevention and control of V. parahaemolyticus infection. Based on the principle of propidium monoazide quantitative PCR (PMA-qPCR) assay to distinguish the live or death of bacteria according to the membrane integrity, the effect of multiple parameters in PMA-qPCR on the viable count was detected and the quantitative detection method of VBNC V.parahaemolyticus was optimized and established. The results indicated that the optimal treatment method was to incubate in dark with 15 mg/L of PMA for 10 min and followed by 15 min of strong light irradiation. Under the optimal conditions, DNA amplification of dead cells was effectively inhibited, and the accuracy of qPCR amplification was higher. The number of viable bacteria was significantly linear correlated with the Ct values of qPCR (R2=0.99). The detection limit of PMA-qPCR standard was from 29 to 2.90×108 CFU/mL, the recovery rate range was 92.97%-99.57%, and the standard deviation was 1.95%. In conclusion, the more accurate and reliable quantitative detection method for VBNC V. parahaemolyticus was established and provides an effective method for further research.

Key words： Vibrio parahaemolyticus; viable bacteria counting; propidium monoazide; qPCR; viable but non-culturable

Cite this article

PENG Linyuan , LIN Hong , WANG Jingxue . Development and optimization of PMA-qPCR for the quantitative detection of viable but non-culturable Vibrio parahaemolyticus[J]. Food and Fermentation Industries, 2022 , 48(1) : 247 -252 . DOI: 10.13995/j.cnki.11-1802/ts.027703

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