Detection of viable Lacticaseibacillus paracasei in fermented milk using propidium monoazide combined with quantitative loop-mediated isothermal amplification. Issue 20 (26th November 2021)

• Record Type: Journal Article
• Authors: Hu, Lianxia; Xue, Yuling; Cui, liru; Zhang, Dong; Feng, Lili; Zhang, Wei; Wang, Shijie
• Is Part Of: FEMS microbiology letters. Volume 368:Issue 20(2021)
• Publication Date: 2021-11-26
• Subjects: viable count -- Lacticaseibacillus paracasei -- PMA-qLAMP -- fermented milk -- probiotics -- propidium monoazide; Microbiology -- Periodicals; 579
• DOI: 10.1093/femsle/fnab148
• Languages: English
• ISSNs: 0378-1097

Abstract

ABSTRACT: To quantify viable probiotic Lacticaseibacillus paracasei ( L. paracasei ) in fermented milk accurately and quickly, propidium monoazide combined with quantitative loop-mediated isothermal amplification (PMA-qLAMP) was applied. The optimal PMA treatment conditions for treating a L. paracasei suspension were determined using an orthogonal test to eliminate the DNA amplification of 10 8 CFU/mL of dead L. paracasei . Primers were designed based on the species-specific gyr B gene of L. paracasei . A phylogenetic tree based on the gyr B gene showed that L. paracasei clustered on the same branch with 91% support. Compared with the 16 strains commonly found in fermented milk, three strains of L. paracasei showed positive PMA-qLAMP results, and the melting temperature was approximately 82.4°C. There was a linear relationship ( R 2 = 0.9983) between the Ct values and the logarithm of the concentration of viable bacteria. The PMA-qLAMP detection limit for the L. paracasei artificially added to fermented milk was 7.3 × 10 2 CFU/mL. There was no significant difference between the logarithm values of the concentration of viable L. paracasei of 50 fermented milk samples within shelf life using the PMA-qLAMP and plate count methods ( P > 0.01). PMA-qLAMP is specific and accurate for obtaining reliable results faster than when using plate counts. Abstract : Quantitative PMA-qLAMP is a specific and accurate method for the detection of viable L. paracasei in fermented milk.