Simultaneous detection of six diarrhea-causing bacterial pathogens with an in-house PCR-luminex assay - PubMed (original) (raw)

doi: 10.1128/JCM.05416-11. Epub 2011 Nov 9.

Jean Gratz, Athanasia Maro, Happy Kumburu, Gibson Kibiki, Mami Taniuchi, Arif Mahmud Howlader, Shihab U Sobuz, Rashidul Haque, Kaisar A Talukder, Shahida Qureshi, Anita Zaidi, Doris M Haverstick, Eric R Houpt

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Simultaneous detection of six diarrhea-causing bacterial pathogens with an in-house PCR-luminex assay

Jie Liu et al. J Clin Microbiol. 2012 Jan.

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.

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Figures

Fig 1

Fig 1

Correlation between template copy numbers and detection by multiplex PCR-Luminex assay (cMFI) for Vibrio cholerae. Best-fit lines and regression (_R_2) were extrapolated. The other bacteria are presented in Fig. S1 in the supplemental material.

Fig 2

Fig 2

Correlation of PCR-Luminex assay cMFI with real-time PCR CT values on clinical samples. Data are shown for fecal samples infected with Aeromonas, Campylobacter, and Salmonella (A) and Shigella and Vibrio (B) that were positive by both PCR-Luminex assay and real-time PCR using the same primers and probe designs.

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