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Updated: Aug 19, 2026
A High-throughput Shigella-specific Bactericidal Assay
Published on: February 27, 2019
Detection of virulence genes in Malaysian Shigella species by multiplex PCR assay
Kwai Lin Thong1, Susan Ling Ling Hoe, S D Puthucheary
1Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. thongkl@um.edu.my
Background:
In Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium and confirmation of the disease by microbiological and biochemical methods remain as the "gold standard". This study aimed to detect the prevalence of four Shigella virulence genes present concurrently, in randomly selected Malaysian strains via a rapid multiplex PCR (mPCR) assay.
Methods:
A mPCR assay was designed for the simultaneous detection of chromosomal- and plasmid-encoded virulence genes (set1A, set1B, ial and ipaH) in Shigella spp. One hundred and ten Malaysian strains (1997-2000) isolated from patients from various government hospitals were used. Reproducibility and sensitivity of the assay were also evaluated. Applicability of the mPCR in clinical settings was tested with spiked faeces following preincubation in brain heart infusion (BHI) broth.
Results:
The ipaH sequence was present in all the strains, while each of the set1A, set1B and ial gene was present in 40% of the strains tested. Reproducibility of the mPCR assay was 100% and none of the non-Shigella pathogens tested in this study were amplified. The mPCR could detect 100 colony-forming units (cfu) of shigellae per reaction mixture in spiked faeces following preincubation.
Conclusions:
The mPCR system is reproducible, sensitive and is able to identify pathogenic strains of shigellae irrespective of the locality of the virulence genes. It can be easily performed with a high throughput to give a presumptive identification of the causal pathogen.
Insights
A rapid multiplex PCR (mPCR) assay effectively detected Shigella virulence genes in Malaysian childhood diarrhea cases. This sensitive and reproducible method offers a high-throughput solution for identifying pathogenic Shigella strains.
Area of Science:
- Microbiology
- Molecular Biology
- Infectious Diseases
Background:
- Shigella spp. is a leading cause of childhood diarrhea in Malaysia.
- Current diagnostic methods rely on traditional microbiological and biochemical techniques.
- There is a need for rapid and accurate detection of Shigella virulence factors.
Purpose of the Study:
- To develop and evaluate a multiplex PCR (mPCR) assay for detecting four key Shigella virulence genes simultaneously.
- To determine the prevalence of these genes in Malaysian Shigella strains.
- To assess the assay's reproducibility, sensitivity, and applicability in clinical settings.
Main Methods:
- A multiplex PCR (mPCR) assay was designed to detect set1A, set1B, ial, and ipaH virulence genes.
- One hundred and ten Malaysian Shigella strains isolated between 1997-2000 were analyzed.
- The assay's performance was validated using spiked fecal samples.
Main Results:
- The ipaH gene was detected in 100% of the strains.
- The set1A, set1B, and ial genes were each present in 40% of the tested strains.
- The mPCR assay demonstrated 100% reproducibility and could detect as few as 100 cfu/mL in spiked feces.
Conclusions:
- The developed mPCR system is a reproducible and sensitive tool for identifying pathogenic Shigella strains.
- This assay allows for high-throughput presumptive identification of Shigella, regardless of virulence gene location.
- The mPCR assay shows promise for rapid clinical diagnostics of Shigella-related diarrhea.
