Affiliation:
1. Yakult Central Institute for Microbiological Research, Kunitachi, Tokyo, Japan
Abstract
ABSTRACT
We developed a PCR-based method to detect and quantify viable
Bifidobacterium bifidum
BF-1 cells in human feces. This method (PMA-qPCR) uses propidium monoazide (PMA) to distinguish viable from dead cells and quantitative PCR using a BF-1-specific primer set designed from the results of randomly amplified polymorphic DNA analysis. During long-term culture (10 days), the number of viable BF-1 cells detected by counting the number of CFU on modified MRS agar, by measuring the ATP contents converted to CFU, and by using PMA-qPCR decreased from about 10
10
to 10
6
cells/ml; in contrast, the total number of (viable and dead) BF-1 cells detected by counting 4′,6-diamidino-2-phenylindolee (DAPI)-stained cells and by using qPCR without PMA and reverse transcription-qPCR remained constant. The number of viable BF-1 cells in fecal samples detected by using PMA-qPCR was highly and significantly correlated with the number of viable BF-1 cells added to the fecal samples, within the range of 10
5.3
to 10
10.3
cells/g feces (wet weight) (
r
> 0.99,
P
< 0.001). After 12 healthy subjects ingested 10
10.3
to 10
11.0
CFU of BF-1 in a fermented milk product daily for 28 days, 10
4.5 � 1.5
(mean � standard deviation [SD]) BF-1 CFU/g was detected in fecal samples by using strain-specific selective agar; in contrast, 10
6.2 � 0.4
viable BF-1 cells/g were detected by using PMA-qPCR, and a total of 10
7.6 � 0.7
BF-1 cells/g were detected by using qPCR without PMA. Thus, the number of viable BF-1 cells detected by PMA-qPCR was about 50 times higher (
P
< 0.01) than that detected by the culture-dependent method. We conclude that strain-specific PMA-qPCR can be used to quickly and accurately evaluate viable BF-1 in feces.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
40 articles.
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