Rapid genetic typing of diarrheagenic Escherichia coli using a two-tube modified molecular beacon based multiplex real-time PCR assay and its clinical application

Abstract

BackgroundDiarrheagenic Escherichia coli (DEC), including Enterotoxigenic E.coli (ETEC), Enteroaggregative E.coli (EAEC), Enteropathogenic E.coli (EPEC), Enterohemolysin E.coli (EHEC) and Enteroinvasive E.coli (EIEC) causes diarrhea or hemolytic uremic syndromes among infants and travelers around the world. A rapid, reliable and repeatable method is urgent for identifying DEC so as to provide the reference for responding to diarrheal disease outbreak and the treatment of the diarrheal patients associated with DEC.MethodsIn this study, specific primers and modified molecular beacon probes of nine specific virulence genes, whose 5′end were added with homo tail sequence, were designed; and a two-tube modified molecular beacon based multiplex real–time PCR (rtPCR) assay for the identification of five Escherichia coli pathotypes, including ETEC, EAEC, EPEC, EHEC and EIEC was developed and optimized. Totally 102 bacterial strains, including 52 reference bacterial strains and 50 clinical strains were detected to confirm whether the target genes selected were specific. Then detection limits of the assay were tested. Lastly, the assay was applied to the detection of 11860 clinical samples to evaluate the specificity and sensitivity of the developed assay compared with the conventional PCR.ResultsThe target genes were 100% specific as assessed on 102 bacterial strains since no cross-reactions were observed. The detection limits ranged from 88 CFU/mL (EHEC) to 880 CFU/mL (EPEC). Compared with the conventional PCR, the specificity and sensitivity of the multiplex rtPCR was 100% and over 99%, respectively. The coefficient of variation (CV) for each target gene ranged from 0.45% to 1.53%. 171 positive clinical samples were mostly identified as ETEC (n = 111, 64.9%) and EPEC (n = 38, 22.2%), which were the dominating pathotypes of DEC strains.ConclusionThe developed multiplex rtPCR assay for the identification of DEC was high sensitive and specific and could be applied to the rapid identification of DEC in clinical and public health laboratories.

The 12 Gastrointestinal Pathogens Spectrum of Acute Infectious Diarrhea in a Sentinel Hospital, Shenzhen, China

“…Similarly, the non-infection of EHEC may benefit from the habit of consumption of cooked food and improvement of hygiene, as most of the EHEC-associated outbreaks was transmitted through contaminated foods (Viazis and Diez-Gonzalez, 2011). It was consistent with our previous surveillance data that EHEC had a very low prevalence (4/11860, 0.03%) in this region (Chen et al, 2014). …”

“…All the V. parahaemolyticus isolates were identified using the Sensititre GNID (TREK Diagnostic System, Cleveland, OH, USA) and serotyped with a commercial serotyping kit (Denka Seiken, Tokyo, Japan), followed by real-time PCR detection of the thermostable direct hemolysin ( tdh ) and thermolabile hemolysin ( trh ) genes (Blackstone et al, 2003; Davis et al, 2004). Stool samples were examined for diarrhoeagenic E. coli by detecting virulence loci of a pool of three suspicious clonies from MacConkey Agar Plate, such as eaeA, stx 1 , and stx 2 of enterohemorrhagic E. coli (EHEC); eaeA and escV of enteropathogenic E. coli (EPEC); ipaH of enteroinvasive E. coli (EIEC) and Shigella strains; lt, sth , and stp of enterotoxigenic E. coli (ETEC); and aggR of enteroaggregative E. coli (EAEC) (Chen et al, 2014). To reduce the risk of false negative results, the multiple PCR method simultaneously detecting five bacterial pathogens associated with diarrhea and the PCR assay targeting DEC were adopted to screening the purified nucleic acid (Chen et al, 2014; Hu et al, 2014).…”

“…Stool samples were examined for diarrhoeagenic E. coli by detecting virulence loci of a pool of three suspicious clonies from MacConkey Agar Plate, such as eaeA, stx 1 , and stx 2 of enterohemorrhagic E. coli (EHEC); eaeA and escV of enteropathogenic E. coli (EPEC); ipaH of enteroinvasive E. coli (EIEC) and Shigella strains; lt, sth , and stp of enterotoxigenic E. coli (ETEC); and aggR of enteroaggregative E. coli (EAEC) (Chen et al, 2014). To reduce the risk of false negative results, the multiple PCR method simultaneously detecting five bacterial pathogens associated with diarrhea and the PCR assay targeting DEC were adopted to screening the purified nucleic acid (Chen et al, 2014; Hu et al, 2014). …”

“…A common tag sequence was added at the 5′ end of each primer to minimum the formation of primer dimer. The amplification reaction was the same as previously described (Chen et al, 2014; Hu et al, 2014). The cycling conditions included a initiating stage of 3 min at 95°C followed by 5 cycles of 10 s at 95°C, 20 s at 58°C, and 15 s at 72°C and finally by a 40 cycles of 10 s at 94°C, 20 s at 55°C, and 15 s at 72°C (Bio-Rad CFX96, Bio-Rad, USA).…”

Investigation of Pathogenic Escherichia coli Strains in Patients with Diarrhea

“…Buna karşın, M-PCR ile amplifiye edilen sırasıyla 376 ve 367 baz çifti (bç) boyutundaki eae ve bfpA genleri ile, sırasıyla 322 ve 320 bç boyutundaki eltB ve ial gen parçalarının büyüklüklerinin birbirine çok yakın olması nedeniyle, sonuçların klasik PCR kullanılarak doğrulanması gerektiği de vurgulanmaktadır 14 . Chen ve arkadaşları 15 ise yaptıkları çalışmada, diyarejenik E.coli'nin tanısında klasik PCR ile karşılaştırıldığında M-PCR'nin özgüllüğünün %100, duyarlılığının %99 olduğunu bildirmişlerdir. Flora bakterileri ile patojen E.coli'lerin ayrılmasındaki güç-lükler nedeniyle, PCR çalışmalarında en azından dört laktoz pozitif ve iki laktoz negatif koloninin çalışmaya alınması veya en az 10 koloni içeren bir sürüntünün petri kutusundan seçilmesinin patojen bakterilerin yakalanma olasılığını artıracağı söylenebilir 9,14 .…”