Real-Time PCR Quantification of Protease-Producing Bacteria in Traditional Chinese Fish Sauce

This study aims to isolate, identify, and quantify protease-producing bacteria in traditional Chinese fish sauce. Fifty-five protease-producing bacteria were isolated from 10 fermented fish sauce samples in five growth media based on their morphology and caseinolytic activity. These isolates were identified through their 16S rDNA gene sequences. BLAST analysis of 16S rDNA sequences revealed that 46 and 9 strains belonged to Bacillus sp. and Virgibacillus halodenitrificans, respectively. We used EvaGreen dye in real-time PCR assay to target the partial bacterial 16S rDNA gene sequences of the 55 strains from fish sauce. Two primer pairs (A and B) were designed. Primer pair B was more suitable than primer pair A for real-time PCR assay, in which the optimum annealing temperature was 60 A degrees C. The significance of the developed method was proven by the highly linear characteristic of the standard curve that relates lower threshold cycle (Ct) values and 16S rDNA copy numbers of the standard DNA sample. This method was used to calculate the number of protease-producing bacteria in fish sauce. The minimum level of detection (1.44 x 10(3) copies/mu L) was also verified. The concentration of protease-producing bacteria in fish sauce was estimated to be (1.47 A +/- 0.73) x 10(3) colony-forming units (cfu)/mL by real-time PCR assay and showed a percentage of positive results correctly assigned of 91.8 % when compared to the plate culture method used as reference. The results may serve as a foundation for future studies on the microbial succession and variation of microorganisms during fish sauce fermentation.