title: 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 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.
authors.affiliation: Division of Infectious Diseases and International Health, Department of Medicine,a and Department of Pathology,e University of Virginia, Charlottesville, Virginia, USA. jl5yj@virginia.edu
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.forename: Jean
authors.initials: J
authors.lastname: Gratz
authors.forename: Athanasia
authors.initials: A
authors.lastname: Maro
authors.forename: Happy
authors.initials: H
authors.lastname: Kumburu
authors.forename: Gibson
authors.initials: G
authors.lastname: Kibiki
authors.forename: Mami
authors.initials: M
authors.lastname: Taniuchi
authors.forename: Arif Mahmud
authors.initials: AM
authors.lastname: Howlader
authors.forename: Shihab U
authors.initials: SU
authors.lastname: Sobuz
authors.forename: Rashidul
authors.initials: R
authors.lastname: Haque
authors.forename: Kaisar A
authors.initials: KA
authors.lastname: Talukder
authors.forename: Shahida
authors.initials: S
authors.lastname: Qureshi
authors.forename: Anita
authors.initials: A
authors.lastname: Zaidi
authors.forename: Doris M
authors.initials: DM
authors.lastname: Haverstick
authors.forename: Eric R
authors.initials: ER
authors.lastname: Houpt
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D003967:Diarrhea / Q000382:microbiology*; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis* / Q000382:microbiology*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
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