Designing primers and evaluation of the efficiency of propidium monoazide – Quantitative polymerase chain reaction for counting the viable cells of Lactobacillus gasseri and Lactobacillus salivarius

• Published in: Yàowu shi͡p︡in fenxi 2017-07, Vol.25 (3), p.533-542
• Main Authors: Lai, Chieh-Hsien, Wu, Sih-Rong, Pang, Jen-Chieh, Ramireddy, Latha, Chiang, Yu-Cheng, Lin, Chien-Ku, Tsen, Hau-Yang
• Format: Article
• Language: English
• Subjects: Azides; Bacteria; Bacterial Load; Bifidobacterium; Counting; Deoxyribonucleic acid; Dietary supplements; Dilution; DNA; DNA Primers; DNA, Bacterial; Efficiency; Enterococcus; Genetic testing; Heat; Lactic acid; Lactobacillus; Lactobacillus gasseri; Lactobacillus salivarius; Microbial Viability; Original; Polymerase Chain Reaction; Primers; Probiotics; Propidium - analogs & derivatives; propidium monoazide; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction; rRNA; rRNA 23S; Spacer; Transcription; viable and heat-killed cells
• ISSN: 1021-9498; 2224-6614
• DOI: 10.1016/j.jfda.2016.10.004

The purpose of this study is to evaluate the efficiency of using propidium monoazide (PMA) real-time quantitative polymerase chain reaction (qPCR) to count the viable cells of Lactobacillus gasseri and Lactobacillus salivarius in probiotic products. Based on the internal transcription spacer and 23S rRNA genes, two primer sets specific for these two Lactobacillus species were designed. For a probiotic product, the total deMan Rogosa Sharpe plate count was 8.65±0.69 log CFU/g, while for qPCR, the cell counts of L. gasseri and L. salivarius were 8.39±0.14 log CFU/g and 8.57±0.24 log CFU/g, respectively. Under the same conditions, for its heat-killed product, qPCR counts for L. gasseri and L. salivarius were 6.70±0.16 log cells/g and 7.67±0.20 log cells/g, while PMA-qPCR counts were 5.33±0.18 log cells/g and 5.05±0.23 log cells/g, respectively. For cell dilutions with a viable cell count of 8.5 log CFU/mL for L. gasseri and L. salivarius, after heat killing, the PMA-qPCR count for both Lactobacillus species was near 5.5 log cells/mL. When the PMA-qPCR counts of these cell dilutions were compared before and after heat killing, although some DNA might be lost during the heat killing, significant qPCR signals from dead cells, i.e., about 4–5 log cells/mL, could not be reduced by PMA treatment. Increasing PMA concentrations from 100 μM to 200 μM or light exposure time from 5 minutes to 15 minutes had no or, if any, only minor effect on the reduction of qPCR signals from their dead cells. Thus, to differentiate viable lactic acid bacterial cells from dead cells using the PMA-qPCR method, the efficiency of PMA to reduce the qPCR signals from dead cells should be notable. [Display omitted] •Counting the viable cells for each strain in a probiotic is important.•Primers specific for L. gasseri and L. salivarius were designed.•The efficiency of PMA-qPCR to count viable LAB needs to be concerned.•Increase of PMA dose or lighting time could not reduce more qPCR signals.