Simultaneous Detection of Six Diarrhea-Causing Bacterial Pathogens with an In-House PCR-Luminex Assay

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 pathogenicAeromonas,Campylobacter jejuni,Campylobacter coli,Salmonella,Shigella, enteroinvasiveEscherichia coli(EIEC),Vibrio, andYersinia. 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 10³to 10⁵CFU/g of stool for each pathogen as well as quantitative detection up to 10⁹CFU/g. A total of 205 clinical fecal specimens from individuals with diarrhea, previously cultured for enteric pathogens and tested forCampylobacterby ELISA, were evaluated. Using these predicate methods as standards, sensitivities and specificities of the PCR-Luminex assay were 89% and 94% forAeromonas, 89% and 93% forCampylobacter, 96% and 95% forSalmonella, 94% and 94% forShigella, 92% and 97% forVibrio, and 100% and 100% forYersinia, 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.