Japanese Journal of Food Microbiology
Online ISSN : 1882-5982
Print ISSN : 1340-8267
Original
Comprehensive Detection of Food-Borne Pathogens from Fecal Specimens Using a Reverse Transcription Multiplex PCR with Fluorescent Dye-Labeled Primers
Naoki SHIGEMOTOYukie TANIZAWAHiroko YAMADASachiko OHARATakeshi MATSUOShinji FUKUDA
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2012 Volume 29 Issue 1 Pages 11-17

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Abstract

We have developed a reverse transcription (RT)-multiplex PCR assay with fluorescent dye-labeled primers for detection of 12 pathogens, including 9 bacteria and 3 viruses, that are highly associated with food-borne gastroenteritis outbreaks. This assay has a great advantage of comprehensive detection of bacterial DNAs and viral RNAs. PCR products of pathogens are easily discriminated by fluorescent color and fragment size visualized under ultraviolet light without staining, after electrophoresis. Simultaneous extraction of bacterial DNAs and viral RNAs were achieved using a commercial viral RNA extraction kit with some modification. After RT reaction, multiplex PCR was carried out with 3 primer sets (A, B, and C) labeled with 3 to 4 different colors of fluorescent dyes. Primer set A was designed for diarrheagenic Escherichia coli. Primer set B was for Salmonella spp., Campylobacter jejuni and C. coli, and Vibrio parahaemolyticus. Primer set C was for Clostridium perfringens, Norovirus, Sapovirus, and Astrovirus. Upon analysis of 45 food-borne gastroenteritis outbreaks and 15 sporadic cases, this assay had nearly corresponding sensitivity and specificity compared with conventional methods, such as bacterial cultivation and monoplex PCR. This assay can be completed within 6 to 7 hours and is considered effective for rapid screening of food-borne bacteria and viruses.

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© 2012 Japanese Society of Food Microbiology
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