Development of Strain-Specific PCR Primers for Quantitative Detection of <i>Bacillus mesentericus</i> Strain TO-A in Human Feces

Abstract

Strain-specific polymerase chain reaction (PCR) primers for detection of Bacillus mesentericus strain TO-A (BM TO-A) were developed. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. A 991-bp RAPD marker found to be strain-specific was sequenced, and two primer pairs specific to BM TO-A were constructed based on this sequence. In addition, we explored a more specific DNA region using inverse PCR, and designed a strain-specific primer set for use in real-time quantitative PCR (qPCR). These primer pairs were tested against 25 Bacillus subtilis strains and were found to be strain-specific. After examination of the detection limit and linearity of detection of BM TO-A in feces, the qPCR method and strain-specific primers were used to quantify BM TO-A in the feces of healthy volunteers who had ingested 3 10 8 colony forming unit (CFU) of BM TO-A per day in tablets. During the administration period, BM TO-A was detected in the feces of all 24 subjects, and the average number of BM TO-A detected using the culture method and qPCR was about 10 4.8 and 10 5.8 cells per gram of feces, respectively. Using the qPCR method, BM TO-A was detected in the feces of half of the subjects 3 d after withdrawal, and was detected in the feces of only one subject 1 week after withdrawal. These results suggest that the qPCR method using BM TO-A strain-specific primers is useful for the quantitative detection of this strain in feces.Key words Bacillus subtilis; strain-specific primer; quantitative polymerase chain reaction (PCR); human feces Probiotics are live microbial food supplements that beneficially affect the host by improving its microbial balance.

Ameliorating effect of probiotics in a rat model of chronic kidney disease

“…It was also reported that B . subtilis TO-A produces bifidobacteria growth factors in the intestine and increases its amount [ 83 ].…”

<i>Bacillus subtilis</i> TO-A extends the lifespan of <i>Caenorhabditis elegans</i>

“…Oral probiotic preparations that contain Clostridium butyricum TO-A (CB), Enterococcus faecium T-110 (EF), and Bacillus subtilis TO-A (BS) exert beneficial effects on the health of hosts, including humans and livestock. Preparations containing these organisms have been utilized as probiotic drugs for treating intestinal diseases such as constipation, diarrhea, irritable bowel syndrome, enteritis, and other infectious diseases for more than 50 years [ 4 , 5 , 6 , 7 , 8 ]. In addition, it was recently suggested that these probiotic bacteria could reduce the risk of recurrent spontaneous preterm delivery in pregnant women [ 9 ].…”