| RRC ID |
29438
|
| 著者 |
Kobayashi A, Sano D, Taniuchi A, Ishii S, Okabe S.
|
| タイトル |
Use of a genetically-engineered Escherichia coli strain as a sample process control for quantification of the host-specific bacterial genetic markers.
|
| ジャーナル |
Appl Microbiol Biotechnol
|
| Abstract |
Quantitative PCR (qPCR) assays targeting the host-specific Bacteroides-Prevotella 16S rRNA genetic markers have been proposed as one of the promising approaches to identify the source of fecal contamination in environmental waters. One of the concerns of qPCR assays to environmental samples is the reliability of quantified values, since DNA extraction followed by qPCR assays are usually performed without appropriate sample process control (SPC) and internal amplification controls (IACs). To check the errors in sample processing and improve the reliability of qPCR results, it is essential to evaluate the DNA recovery efficiency and PCR amplification efficiency of the target genetic markers and correct the measurement results. In this study, we constructed a genetically-engineered Escherichia coli K12 strain (designated as strain MG1655 Δlac::kan) as sample process control and evaluated the applicability to environmental water samples. The recovery efficiency of the SPC strain MG1655 Δlac::kan was similar to that of Bacteroides fragilis JCM 11019, when DNA were extracted from water samples spiked with the two bacteria. Furthermore, the SPC was included in the qPCR assays with propidium monoazide (PMA) treatment, which can exclude the genetic markers from dead cells. No significant DNA loss was observed in the PMA treatment. The inclusion of both the SPC (strain MG1655 Δlac::kan) and IAC in qPCR assays with PMA treatment gave the assurance of reliable results of host-specific Bacteroides-Prevotella 16S rRNA genetic markers in environmental water samples.
|
| 巻・号 |
97(20)
|
| ページ |
9165-73
|
| 公開日 |
2013-10-1
|
| DOI |
10.1007/s00253-013-5188-z
|
| PMID |
23989919
|
| MeSH |
Animals
Bacteroides / genetics
Bacteroides / isolation & purification*
Bacteroides / physiology
Cattle
Chickens
Escherichia coli / genetics*
Escherichia coli / isolation & purification
Escherichia coli / metabolism
Escherichia coli Proteins / genetics
Escherichia coli Proteins / metabolism
Feces / microbiology*
Fresh Water / chemistry
Fresh Water / microbiology*
Genetic Engineering
Genetic Markers
Host Specificity
Humans
Polymerase Chain Reaction / methods
Polymerase Chain Reaction / standards*
Prevotella / genetics
Prevotella / isolation & purification*
Prevotella / physiology
Quality Control
Species Specificity
Swine
Waste Water / chemistry
Waste Water / microbiology*
|
| IF |
3.53
|
| 引用数 |
8
|
|
WOS 分野
|
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
|
| リソース情報 |
| 一般微生物 |
JCM 11019 |
