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Evaluation of Multiplex Pcr System for Simultaneous Detection of Escherichia Coli O157:h7, Listeria Monocytogenes and Salmonella Enteritidis in Shrimp Samples

Mahmuda Yasmin
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh.

Susumu Kawasaki
National Food Research Institute, Food Hygiene Team, Kanondai-2-1-12, Tsukuba, 305-8642, Japan.

Shinichi Kawamoto
National Food Research Institute, Food Hygiene Team, Kanondai-2-1-12, Tsukuba, 305-8642, Japan.

Abstract/ Executive Summary:

A multiplex polymerase chain reaction (PCR) method was evaluated for simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enteritidis in shrimp samples. The sensitivity of DNA amplification by PCR in this method was found to be 10³cfu/ml for each pathogen. When this protocol was adopted for the detection of each of the above mentioned pathogen in spiked shrimp extract culture, similar sensitivity was observed. However, this method detected 1 bacterial cell for E. coli O157:H7 and S. enteritidis and 100 for L. monocytogenes per 25 g spiked shrimp samples after overnight enrichment. In the commercially imported shrimp samples, none was found to contain any of the three pathogens by multiplex PCR or by conventional method, which suggests that the multiplex PCR is a reliable and useful for rapid screening of shrimp samples for E. coli O157:H7, L. monocytogenes and S. enteritidis. This will save time and increase our ability to assure food safety.

Keywords: Multiples PCR, Shrimp extract, Spiked sample, Escherichia coli O157:H7, Listeria monocytogenes

Location:

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Shrimp

Objectives:

To evaluate the optimization of the pre-enrichment conditions, the DNA extraction method, and the multiplex PCR settings.

Methodology:

Bacterial strains and culture conditions: The bacterial strains used in this experiment were from our laboratory collection, which included L. monocytogenes strains ATCC 43256 and ATCC 49594, S. enteritidis IFO 3313 and E. coli O157:H7. The strains were maintained tryptic soy agar (TSA; Nissui Seiaku Ltd., Tokyo, Japan) slants at 4^oC. Bacterial stains were usually grown in trypticase soy broth (TSB; BBL, Becton Dickinson, Sparks, USA) at 37^oC overnight. For viable cell determination, serial decimal dilutions of samples with phosphate buffered saline (PBS, pH 7.2) were made and plated on deoxycholate hydrogen sulphide lactose agar (Eiken Co., Ltd., Tokyo, Japan) for Salmonella, on Oxoid modified agar (Oxoid, Hampshire, UK) for L. monocytogenes, and on modified E. coli agar (Eiken Co., Ltd., Tokyo, Japan) for E. coli O157:H7. The plates were then inoculated at 37^oC for 24 to 48 h before the colonies were counted.

Source of Information: Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 42-46

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In the commercially imported shrimp samples, none was found to contain any of the three pathogens by multiplex PCR or by conventional method, which suggests that the multiplex PCR is a reliable and useful for rapid screening of shrimp samples for E. coli O157:H7, L. monocytogenes and S. enteritidis. This will save time and increase our ability to assure food safety.

Knowledge Management: Journal