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Published on: November 7, 2018
Simultaneous detection of six diarrhea-causing bacterial pathogens with an in-house PCR-luminex assay
Jie Liu1, Jean Gratz, Athanasia Maro
1Division of Infectious Diseases and International Health, Department of Medicine,a and Department of Pathology,e University of Virginia, Charlottesville, Virginia, USA. jl5yj@virginia.edu
Abstract:
Diarrhea can be caused by a range of pathogens, including several bacteria. Conventional diagnostic methods, such as culture, biochemical tests, and enzyme-linked immunosorbent assay (ELISA), are laborious. We developed a 7-plex PCR-Luminex assay to simultaneously screen for several of the major diarrhea-causing bacteria directly in fecal specimens, including pathogenic Aeromonas, Campylobacter jejuni, Campylobacter coli, Salmonella, Shigella, enteroinvasive Escherichia coli (EIEC), Vibrio, and Yersinia. We included an extrinsic control to verify extraction and amplification. The assay was first validated with reference strains or isolates and exhibited a limit of detection of 10(3) to 10(5) CFU/g of stool for each pathogen as well as quantitative detection up to 10(9) CFU/g. A total of 205 clinical fecal specimens from individuals with diarrhea, previously cultured for enteric pathogens and tested for Campylobacter by ELISA, were evaluated. Using these predicate methods as standards, sensitivities and specificities of the PCR-Luminex assay were 89% and 94% for Aeromonas, 89% and 93% for Campylobacter, 96% and 95% for Salmonella, 94% and 94% for Shigella, 92% and 97% for Vibrio, and 100% and 100% for Yersinia, respectively. All discrepant results were further examined by singleplex real-time PCR assays targeting different gene regions, which revealed 89% (55/62 results) concordance with the PCR-Luminex assay. The fluorescent signals obtained with this approach exhibited a statistically significant correlation with the cycle threshold (C(T)) values from the cognate real-time PCR assays (P < 0.05). This multiplex PCR-Luminex assay enables sensitive, specific, and quantitative detection of the major bacterial causes of gastroenteritis.
Insights
A new 7-plex PCR-Luminex assay offers rapid, simultaneous detection of major bacterial diarrhea pathogens directly from stool. This method provides sensitive and specific results, improving upon traditional diagnostic techniques for bacterial gastroenteritis.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Diarrhea is commonly caused by bacterial pathogens.
- Conventional diagnostic methods like culture and ELISA are time-consuming and laborious.
- There is a need for faster, more efficient diagnostic tools for bacterial gastroenteritis.
Purpose of the Study:
- To develop and validate a multiplex PCR-Luminex assay for simultaneous detection of major diarrhea-causing bacteria.
- To compare the performance of the new assay with conventional methods.
Main Methods:
- A 7-plex PCR-Luminex assay was designed to detect Aeromonas, Campylobacter (jejuni and coli), Salmonella, Shigella, enteroinvasive Escherichia coli (EIEC), Vibrio, and Yersinia.
- The assay was validated using reference strains and 205 clinical fecal specimens.
- An extrinsic control was included to monitor extraction and amplification efficiency.
Main Results:
- The assay demonstrated limits of detection from 10^3 to 10^5 CFU/g and quantitative detection up to 10^9 CFU/g.
- High sensitivities and specificities were achieved for all targeted pathogens, with values ranging from 89% to 100%.
- Concordance with singleplex real-time PCR assays was high (89%), and fluorescent signals correlated significantly with C(T) values.
Conclusions:
- The developed multiplex PCR-Luminex assay is a sensitive, specific, and quantitative tool for diagnosing bacterial gastroenteritis.
- This assay offers a significant improvement over conventional methods, enabling simultaneous screening of multiple pathogens.
- The assay facilitates rapid and accurate identification of key bacterial causes of diarrhea.
