2007 Fiscal Year Final Research Report Summary
Development of a Quantitative Real-time Polymerase Chain Reaction Method to Enumerate Total Bacterial Count in Ready-to- eat Fruits and Vegetables
| Project/Area Number |
18580314
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied veterinary science
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| Research Institution | National Institute of Health Sciences |
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Principal Investigator |
YUKIKO Kodo National Institute of Health Sciences, Div. Microbiology, Chief (50218632)
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| Project Period (FY) |
2006 – 2007
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| Keywords | Food / Hygiene / Bacterium / Microbiology / Veterinary |
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| Research Abstract |
A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminating ready-to-eat vegetables and fruits as compared to the standard plate count method. Primers targeting the rpoB gene that encodes for the β-subunit of the bacterial RNA polymerase, common to most bacterial species was used instead of the 16S rRNA gene that has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in wide-range of bacterial species by using 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them using the real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle numbers when compared to the plate count culture number. Further, the predominant bacterial flora detected by the culture method was compared with those detected by the real-time PCR assay using a denaturing gradient gel electrophoresis analysis. The results from the two methods were almost identical. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.
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