title: Development of strain-specific PCR primers for quantitative detection of Bacillus mesentericus 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.
authors.affiliation: Bioscience R&D Department, TOA Pharmaceutical Co., Ltd.
authors.forename: Naoki
authors.initials: N
authors.lastname: Sato
authors.forename: Genichiro
authors.initials: G
authors.lastname: Seo
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Benno
mesh_terms: D001407:Bacillus / Q000235:genetics*; D003106:Colon / Q000382:microbiology*; D015169:Colony Count, Microbial; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D005243:Feces / Q000382:microbiology*; D005819:Genetic Markers; D006801:Humans; D019105:Random Amplified Polymorphic DNA Technique; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D005819:Genetic Markers