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Detection of diarrhoeagenic Escherichia coli in clinical and environmental water sources in South Africa using single-step 11-gene m-PCR.

Omar KB, Barnard TG - World J. Microbiol. Biotechnol. (2014)

Bottom Line: Interestingly, 25 % of the E. coli astA toxin detected in environmental isolates and 17 % in clinical isolates did not contain any of the other virulence genes tested.In conclusion, the optimised single-step 11-gene m-PCR reactions could be successfully used for the identification of pathogenic and non-pathogenic E. coli types.The m-PCR was also successful in showing monitoring for PCR inhibition to ensure correct reporting of the results.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Health Sciences, Water and Health Research Centre, University of Johannesburg, Doornfontein, PO Box 17011, Johannesburg, 2028, South Africa, kousaro@uj.ac.za.

ABSTRACT
Escherichia coli (E. coli) consists of commensal (ComEC) and diarrhoeagenic (DEC) groups. ComEC are detected using traditional culture methods. Conformational steps are performed after culturing if it is required to test for the presence of DEC, increasing cost and time in obtaining the results. The aim of this study was to develop a single-step multiplex polymerase chain reaction (m-PCR) that can simultaneously amplify genes associated with DEC and ComEC, with the inclusion of controls to monitor inhibition. A total of 701 samples, taken from clinical and environmental water sources in South Africa, were analysed with the optimised m-PCR which targeted the eaeA, stx1, stx2, lt, st, ial, eagg, astA and bfp virulence genes. The mdh and gapdh genes were included as an internal and external control, respectively. The presence of the external control gapdh gene in all samples excluded any possible PCR inhibition. The internal control mdh gene was detected in 100 % of the environmental and 85 % of the clinical isolates, confirming the classification of isolates as E. coli PCR positive samples. All DEC types were detected in varying degrees from the mdh positive environmental and clinical isolates. Important gene code combinations were detected for clinical isolates of 0.4 % lt and eagg. However, 2.3 % of eaeA and ial, and 8.7 % of eaeA and eagg were reported for environmental water samples. The E. coli astA toxin was detected as positive at 35 and 17 % in environmental isolates and clinical isolates, respectively. Interestingly, 25 % of the E. coli astA toxin detected in environmental isolates and 17 % in clinical isolates did not contain any of the other virulence genes tested. In conclusion, the optimised single-step 11-gene m-PCR reactions could be successfully used for the identification of pathogenic and non-pathogenic E. coli types. The m-PCR was also successful in showing monitoring for PCR inhibition to ensure correct reporting of the results.

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Related in: MedlinePlus

Agarose gel of the PCR products obtained for the E. coli multiplex PCR (lane2). No template control (NTC) in (lane 2). The molecular weight marker is shown in (lane 1)
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Fig1: Agarose gel of the PCR products obtained for the E. coli multiplex PCR (lane2). No template control (NTC) in (lane 2). The molecular weight marker is shown in (lane 1)

Mentions: The main challenge of designing a multiplex PCR is the possibility of primer dimers and non-specific results which is a risk for false positive and negative results. Therefore, it is necessary to design and include primers with close annealing temperatures and to begin the program with a hotstart as reported by Vidal et al. (2005). The effect of the wide temperature range is overcome by the addition of Q-solution that is supplied by the manufacturer and that can be included with the enzyme. A wide variety of temperatures were tested before the final version of the multiplex PCR was optimized and tested. The results confirm that the single m-PCR was successfully compiled to detect all of the targeted genes in a single reaction even though primers with different melting temperatures ranging from 50 to 73 °C were used (Fig. 1). The PCR amplicons were confirmed as the correct target gene by sequencing (data not shown) showing the specific amplification of the genes in a mixture of DEC. Fig. 1


Detection of diarrhoeagenic Escherichia coli in clinical and environmental water sources in South Africa using single-step 11-gene m-PCR.

Omar KB, Barnard TG - World J. Microbiol. Biotechnol. (2014)

Agarose gel of the PCR products obtained for the E. coli multiplex PCR (lane2). No template control (NTC) in (lane 2). The molecular weight marker is shown in (lane 1)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4150989&req=5

Fig1: Agarose gel of the PCR products obtained for the E. coli multiplex PCR (lane2). No template control (NTC) in (lane 2). The molecular weight marker is shown in (lane 1)
Mentions: The main challenge of designing a multiplex PCR is the possibility of primer dimers and non-specific results which is a risk for false positive and negative results. Therefore, it is necessary to design and include primers with close annealing temperatures and to begin the program with a hotstart as reported by Vidal et al. (2005). The effect of the wide temperature range is overcome by the addition of Q-solution that is supplied by the manufacturer and that can be included with the enzyme. A wide variety of temperatures were tested before the final version of the multiplex PCR was optimized and tested. The results confirm that the single m-PCR was successfully compiled to detect all of the targeted genes in a single reaction even though primers with different melting temperatures ranging from 50 to 73 °C were used (Fig. 1). The PCR amplicons were confirmed as the correct target gene by sequencing (data not shown) showing the specific amplification of the genes in a mixture of DEC. Fig. 1

Bottom Line: Interestingly, 25 % of the E. coli astA toxin detected in environmental isolates and 17 % in clinical isolates did not contain any of the other virulence genes tested.In conclusion, the optimised single-step 11-gene m-PCR reactions could be successfully used for the identification of pathogenic and non-pathogenic E. coli types.The m-PCR was also successful in showing monitoring for PCR inhibition to ensure correct reporting of the results.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Health Sciences, Water and Health Research Centre, University of Johannesburg, Doornfontein, PO Box 17011, Johannesburg, 2028, South Africa, kousaro@uj.ac.za.

ABSTRACT
Escherichia coli (E. coli) consists of commensal (ComEC) and diarrhoeagenic (DEC) groups. ComEC are detected using traditional culture methods. Conformational steps are performed after culturing if it is required to test for the presence of DEC, increasing cost and time in obtaining the results. The aim of this study was to develop a single-step multiplex polymerase chain reaction (m-PCR) that can simultaneously amplify genes associated with DEC and ComEC, with the inclusion of controls to monitor inhibition. A total of 701 samples, taken from clinical and environmental water sources in South Africa, were analysed with the optimised m-PCR which targeted the eaeA, stx1, stx2, lt, st, ial, eagg, astA and bfp virulence genes. The mdh and gapdh genes were included as an internal and external control, respectively. The presence of the external control gapdh gene in all samples excluded any possible PCR inhibition. The internal control mdh gene was detected in 100 % of the environmental and 85 % of the clinical isolates, confirming the classification of isolates as E. coli PCR positive samples. All DEC types were detected in varying degrees from the mdh positive environmental and clinical isolates. Important gene code combinations were detected for clinical isolates of 0.4 % lt and eagg. However, 2.3 % of eaeA and ial, and 8.7 % of eaeA and eagg were reported for environmental water samples. The E. coli astA toxin was detected as positive at 35 and 17 % in environmental isolates and clinical isolates, respectively. Interestingly, 25 % of the E. coli astA toxin detected in environmental isolates and 17 % in clinical isolates did not contain any of the other virulence genes tested. In conclusion, the optimised single-step 11-gene m-PCR reactions could be successfully used for the identification of pathogenic and non-pathogenic E. coli types. The m-PCR was also successful in showing monitoring for PCR inhibition to ensure correct reporting of the results.

Show MeSH
Related in: MedlinePlus