The DOI matches its Crossref record; 43 of the 43 references it cites were found in a registry or an index.
Sensitive Quantitative Detection of Commensal Bacteria by rRNA-Targeted Reverse Transcription-PCR
Open accessAbstract
A sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) method was developed for exact and sensitive enumeration of subdominant bacterial populations. Using group- or species-specific primers for 16S or 23S rRNA, analytical curves were constructed for Escherichia coli , Enterococcus faecalis , Staphylococcus aureus , Clostridium perfringens , and Pseudomonas aeruginosa , and the threshold cycle value was found to be linear up to an RNA amount of 10 −3 cell per RT-PCR. The number of bacteria in culture was determined by RT-qPCR, and the results correlated well with the CFU count over the range from 10 0 to 10 5 CFU. The bacterial counts obtained by RT-qPCR were the same as the CFU counts irrespective of the growth phase in vitro, except for C. perfringens during starvation periods; the viable cell counts obtained by using a combination of 4′,6-diamidino-2-phenylindole (DAPI) staining and SYTO9-propidium iodide double staining were in good agreement with the RT-qPCR counts rather than with the CFU counts. The RT-qPCR method could detect endogenous Enterobacteriaceae and P. aeruginosa in feces of hospitalized patients ( n = 38) at a level of 10 3 cells per g of feces, and for enumeration of S. aureus or P. aeruginosa spiked into human peripheral blood, the lower detection limit for RT-qPCR quantification of the bacteria was 2 cells per ml of blood, suggesting that this method was equivalent to the conventional culture method. As only 5 h was needed for RT-qPCR quantification, we suggest that rRNA-targeted RT-qPCR assays provide a sensitive and convenient system for quantification of commensal bacteria and for examining their possible invasion of a host.
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Cites
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Cited by
Odamaki, T., Kato, K., Sugahara, H., Hashikura, N., Takahashi, S., Xiao, J.-Z., Abe, F., & Osawa, R. (2016) · BMC Microbiology · doi:10.1186/s12866-016-0708-5 · Cited by 1,521
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Intestinal dysbiosis and lowered serum lipopolysaccharide-binding protein in Parkinson's disease
Hasegawa, S., Goto, S., Tsuji, H., Okuno, T., Asahara, T., Nomoto, K., Shibata, A., Fujisawa, Y., Minato, T., Okamoto, A., et al. (2015) · PLOS ONE · doi:10.1371/journal.pone.0142164 · Cited by 523
Self-citationEstablishment and development of intestinal microbiota in preterm neonates
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Metadata
- Authors
- Matsuda, K., Tsuji, H., Asahara, T., Kado, Y., & Nomoto, K.
- Venue
- Applied and Environmental Microbiology
- Publisher
- American Society for Microbiology
- Published
- 2007-01
- Volume
- 73
- Issue
- 1
- Pages
- 32-39
- ISSN
- 0099-2240 (ISSN-L)
- 1098-5336
- OpenAlex
- W2143366420
- Cited by
- 294
- References
- 43
- FWCI
- 3.68
- Type
- journal article
- Language
- en
- Open access
- green
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