Quantitative Detection of Viable but Nonculturable Using Photosensitive Nucleic Acid Dye PMA Combined with Isothermal Amplification LAMP in Raw Milk.

An accurate method that rapidly detects the number of viable but nonculturable (VBNC) was developed by combining propidium bromide with quantitative LAMP (PMA-QLAMP). The gene was the target for primers design. The optimal PMA treatment conditions were determined to eliminate the DNA amplification of 10 CFU/mL of dead without affecting any viable DNA amplification. Compared with the DNA of 24 strains of common non- strains found in raw milk and dairy products, the DNA of only six strains from different sources was amplified using PMA-QLAMP. The ability of PMA-QLAMP to quantitatively detect non-dead in a 10% powdered infant formula (PIF) solution was limited to 4.3 × 10 CFU/mL and above concentrations. Pasteurizing 10 CFU/mL viable yielded the maximum ratio of the VBNC . PMA-QLAMP-based detection indicated that, although approximately 13% of 60 samples were positive for viable the titers in these positive samples were low, and none entered the VBNC state under pasteurization. PMA-QLAMP showed potential as a specific and reliable method for detecting VBNC- in pasteurized raw milk, thereby providing an early warning system that indicates potential contamination of PIF.