Propidium monoazide based real-time PCR to detect Vibrio parahaemolyticus in a viable but nonculturable state

Abstract:

[Background] Vibrio parahaemolyticus, which is highly pathogenic and harmful to public health, is a common contaminating microorganism in chilled food and meat products. To keep them fresh, imported and exported food are often refrigerated or frozen to prevent microorganism growth during transportation and machining. However, residual V. parahaemolyticus can enter a viable but non-culturable (VBNC) state, which poses potential risks. [Objective] This study aimed to establish a method to detect V. parahaemolyticus in a VBNC state in frozen food and to explore its applicability. [Methods] A solution containing V. parahaemolyticus was added to homogenized Atlantic salmon matrix to a final concentration of 6.6×105 colony forming units (CFU)/mL. Aliquots of the mixture were induced for 10, 20, 30, and 50 days at ?20 °C. A propidium monoazide (PMA)-based quantitative real-time PCR (qPCR) method was established to detect the V. parahaemolyticus in the samples of different frozen periods and compared with the results of qPCR and plate culture methods. [Results] The established PMA-qPCR method showed good specificity and no cross-reaction with other negative reference strains. The sensitivity of the method was also high and the quantitative limit was 19.8 CFU/mL. The variation coefficients (CV) of Cq values were all below 1.5%. The standard curve was y=?3.272x+45.310 and the linear regression coefficient, R2 was 0.996. The quantitative range was 1×102 to 1×109 CFU/mL. After the low temperature induction from 10?50 d, the Cq value of the qPCR method was between 26.32 and 27.34 which showed little change. However, the Cq value of the PMA-qPCR increased from 26.43 to 38.84, showing a significant increase in the number of dead bacteria. After comparison and statistical analyses, the number of live bacteria detected by PMA-qPCR was higher than that of plate culture, and the difference was significant (P<0.05). [Conclusion] The established PMA-qPCR method had high specificity and sensitivity and could effectively inhibit the amplification of dead bacteria. It was also an effective and fast method to detect VBNC bacteria, which could overcome the limitations of the traditional plate culture method.