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Detection of enterotoxigenic Escherichia coli, Shigella and Campylobacter spp. by multiplex PCR assay
B A Oyofo1, Z S Mohran, S H el-Etr
1Enteric Microbiology Branch, U.S. Naval Medical Research Unit No. 3, Cairo, Egypt. namru3@centcom.dsaa.osd.mil
Abstract:
Three oligonucleotide primers were used in a polymerase chain reaction (PCR) assay for the simultaneous amplification of regions of the invasive plasmid antigen (ipaH) of Shigella spp., flagellin gene (flaA) of Campylobacter spp., and heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC). The multiplex assay was performed using DNA extracted by a chaotropic method directly from diarrhoeal stools. The diagnostic efficacy of the assay was analyzed by agarose gel electrophoresis. This assay shows a novel approach for the diagnosis of diarrhoea caused by Shigella spp., ETEC, and Campylobacter spp.
Insights
This study introduces a novel multiplex polymerase chain reaction (PCR) assay for rapid diagnosis of diarrheal diseases. The assay simultaneously detects Shigella spp., enterotoxigenic Escherichia coli (ETEC), and Campylobacter spp. directly from stool samples.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Diarrheal diseases pose a significant global health burden.
- Accurate and rapid identification of causative pathogens is crucial for effective treatment.
- Current diagnostic methods can be time-consuming or lack multiplexing capabilities.
Purpose of the Study:
- To develop and evaluate a novel multiplex polymerase chain reaction (PCR) assay.
- To enable simultaneous detection of three common bacterial causes of diarrhea: Shigella spp., enterotoxigenic Escherichia coli (ETEC), and Campylobacter spp.
- To utilize DNA extracted directly from diarrheal stools for improved diagnostic efficiency.
Main Methods:
- Development of a multiplex PCR assay using three specific oligonucleotide primers.
- Simultaneous amplification of invasive plasmid antigen (ipaH) gene for Shigella spp.
- Amplification of flagellin gene (flaA) for Campylobacter spp. and heat-labile enterotoxin (LT) gene for ETEC.
- DNA extraction from diarrheal stools using a chaotropic method.
- Analysis of diagnostic efficacy via agarose gel electrophoresis.
Main Results:
- Successful simultaneous amplification of target genes from Shigella spp., ETEC, and Campylobacter spp.
- Demonstration of the assay's ability to detect multiple pathogens in a single reaction.
- Validation of DNA extraction method for direct stool sample analysis.
- Agarose gel electrophoresis confirmed the presence and specificity of amplified DNA fragments.
Conclusions:
- The developed multiplex PCR assay offers a novel and efficient approach for diagnosing diarrheal infections.
- This method allows for the simultaneous identification of Shigella spp., ETEC, and Campylobacter spp.
- The assay provides a potentially faster and more comprehensive diagnostic tool for clinical settings.