Abstract: Abstract: Lactobacillus kefiranofaciens subsp. kefiranofaciens is a new food raw material approved in China in 2021, which has broad application prospects in the fields of food, medicine and cosmetics. In order to be able to rapidly and specifically detect the subspecies of Lactobacillus kefiranofaciens subsp. kefiranofaciens, a specific real-time PCR detection method was established. Specific primers were designed to screen specific primers according to the 16S rDNA sequence and whole genome sequence of the ZW3 subspecies of Lactobacillus kefiranofaciens subsp. kefiranofaciens of the model strain, and a real-time PCR method was established by SYBR Green I fluorescent dye, and the specificity, sensitivity, repeatability and mixed system of the method were detected. The results showed that the method established in this study had strong specificity, high sensitivity and good repeatability, and the standard curve of real-time PCR is established, and its linear correlation coefficient R2 is 0.965, which had a good linear relationship. Moreover, this method can be specifically detected in the subspecies and mixed systems of Lactobacillus kefiranofaciens subsp. kefiranofaciens. In summary, the real-time PCR method established in this study could quickly and accurately detect the subspecies of Lactobacillus kefiranofaciens subsp. kefiranofaciens, which provided a new method for the specific qualitative and quantitative detection of Lactobacillus kefiranofaciens subsp. kefiranofaciens.

Key words: Lactobacillus kefiranofaciens subsp. kefiranofaciens, real-time fluorescent quantitative PCR, specific primers