Development of rapid and highly specific TaqMan probe-based real-time PCR assay for the identification and enumeration of Lactobacillus kefiri in kefir milk

Abstract

Lactobacillus kefiri is one of the key functional lactic acid bacteria in kefir milk. We designed a novel real-time PCR primer/probe set, LK_508 targeting the recA gene, for the rapid identification and enumeration of L. kefiri. In inclusivity and exclusivity test using standard strains and kefir isolates, only the 9 tested L. kefiri strains were positive, with the remaining 38 closely related microorganisms testing negative, thus indicating 100% sensitivity and specificity of the assay. The population of L kefiri was 3.77, 4.30, 4.79, and 5.63 log cfu mL(-1) of kefir milk fermented at 25 degrees C with 5% grain-milk ratio for 12, 24, 36, and 48 h, respectively. The newly developed qPCR assay could be applied to investigate the quantitative relationship of kefir microbiota in fermentation process. (C) 2016 Elsevier Ltd. All rights reserved.