RRC ID 27484
著者 Ishii S, Segawa T, Okabe S.
タイトル Simultaneous quantification of multiple food- and waterborne pathogens by use of microfluidic quantitative PCR.
ジャーナル Appl Environ Microbiol
Abstract The direct quantification of multiple pathogens has been desired for diagnostic and public health purposes for a long time. In this study, we applied microfluidic quantitative PCR (qPCR) technology to the simultaneous detection and quantification of multiple food- and waterborne pathogens. In this system, multiple singleplex qPCR assays were run under identical detection conditions in nanoliter-volume chambers that are present in high densities on a chip. First, we developed 18 TaqMan qPCR assays that could be run in the same PCR conditions by using prevalidated TaqMan probes. Specific and sensitive quantification was achieved by using these qPCR assays. With the addition of two previously validated TaqMan qPCR assays, we used 20 qPCR assays targeting 10 enteric pathogens, a fecal indicator bacterium (general Escherichia coli), and a process control strain in the microfluidic qPCR system. We preamplified the template DNA to increase the sensitivity of the qPCR assays. Our results suggested that preamplification was effective for quantifying small amounts of the template DNA without any major impact on the sensitivity, efficiency, and quantitative performance of qPCR. This microfluidic qPCR system allowed us to detect and quantify multiple pathogens from fecal samples and environmental water samples spiked with pathogens at levels as low as 100 cells/liter. These results suggest that the routine monitoring of multiple pathogens in food and water samples is now technically feasible. This method may provide more reliable information for risk assessment than the current fecal contamination indicator approach.
巻・号 79(9)
ページ 2891-8
公開日 2013-5-1
DOI 10.1128/AEM.00205-13
PII AEM.00205-13
PMID 23435884
PMC PMC3623133
MeSH Bacteria / genetics Bacteria / isolation & purification* DNA Primers / genetics DNA, Bacterial / genetics Environmental Monitoring / methods* Escherichia coli / genetics Escherichia coli / isolation & purification Feces / microbiology Food Microbiology* Microfluidics / methods* Oligonucleotide Array Sequence Analysis Plasmids / genetics Polymerase Chain Reaction / methods* Sensitivity and Specificity Water Microbiology*
IF 4.016
引用数 60
WOS 分野 BIOTECHNOLOGY & APPLIED MICROBIOLOGY MICROBIOLOGY
リソース情報
一般微生物 JCM 1290 JCM 1652 JCM 2013 JCM 2529 JCM 6977 JCM 6978 JCM 7571 JCM 7671 JCM 7672 JCM 7673 JCM 7674 JCM 7675 JCM 7676 JCM 7677 JCM 7678 JCM 7679 JCM 7680 JCM 14870