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Detection and identification of enterohemorrhagic Escherichia coli O157:H7 and Vibrio cholerae O139 using oligonucleotide microarray.

Jin DZ, Xu XJ, Chen SH, Wen SY, Ma XE, Zhang Z, Lin F, Wang SQ - Infect. Agents Cancer (2007)

Bottom Line: Multiplex PCR did not produce any non-specific amplicons when 149 related species or genera of standard bacteria were tested (100% specificity).Using recombinant plasmid and target pathogens, we were able to detect positive hybridization signals with 102 copies/muL and 103 cfu/mL per reaction.This assay was a specific and sensitive tool for simultaneous detection and identification of the new subtype of two pathogens causing diarrhea in human.

View Article: PubMed Central - HTML - PubMed

Affiliation: Beijing Institute of Radiation Medicine, Beijing, 100850, China. dazhijin@163.com

ABSTRACT

Background: The rapid and accurate detection and identification of the new subtype of the pathogens is crucial for diagnosis, treatment and control of the contagious disease outbreak. Here, in this study, an approach to detect and identify Escherichia coli O157:H7 and Vibrio cholerae O139 was established using oligonucleotide microarray. We coupled multiplex PCR with oligonucleotide microarray to construct an assay suitable for simultaneous identification of two subtypes of the pathogens.

Results: The stx1, stx2 gene and uidA gene having the specific mutant spot were chosen as the targets for Escherichia coli O157:H7, and meanwhile the ctxA, tcpA, and LPSgt gene for Vibrio cholerae O139. The oligonucleotide microarray was composed of eight probes including negative control and positive control from 16S rDNA gene. The six primers were designed to amplify target fragments in two triplex PCR, and then hybridized with oligonucleotide microarray. An internal control would be to run a PCR reaction in parallel. Multiplex PCR did not produce any non-specific amplicons when 149 related species or genera of standard bacteria were tested (100% specificity). In addition, Escherichia coli O157:H7 and Escherichia coli O157:non-H7, Vibrio cholerae O139 and Vibrio cholerae O1 had been discriminated respectively. Using recombinant plasmid and target pathogens, we were able to detect positive hybridization signals with 102 copies/muL and 103 cfu/mL per reaction.

Conclusion: The DNA microarray assay reported here could detect and identify Escherichia coli O157:H7 and Vibrio cholerae O139, and furthermore the subtype was distinguished. This assay was a specific and sensitive tool for simultaneous detection and identification of the new subtype of two pathogens causing diarrhea in human.

No MeSH data available.


Related in: MedlinePlus

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Mentions: The triplex PCR was used to amplify the target gene segments in E. coli O157:H7 and Vibrio cholerae O139. In multiplex asymmetric PCR reaction, the variation of the ratio of forward primer to reverse primer and concentrations impacted the amplification efficiency obviously. Therefore, the signal intensities of hybridization were highly related to the ratio and amount of forward primer to reverse fluorescent primer for each gene. After the optimization, three PCR products in one tube had good specificity and average amplification efficiency. The concentration of primers was showed in Table 1, and then the result of agarose gel electrophoresis was shown in Figure 1.


Detection and identification of enterohemorrhagic Escherichia coli O157:H7 and Vibrio cholerae O139 using oligonucleotide microarray.

Jin DZ, Xu XJ, Chen SH, Wen SY, Ma XE, Zhang Z, Lin F, Wang SQ - Infect. Agents Cancer (2007)

© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267443&req=5

Mentions: The triplex PCR was used to amplify the target gene segments in E. coli O157:H7 and Vibrio cholerae O139. In multiplex asymmetric PCR reaction, the variation of the ratio of forward primer to reverse primer and concentrations impacted the amplification efficiency obviously. Therefore, the signal intensities of hybridization were highly related to the ratio and amount of forward primer to reverse fluorescent primer for each gene. After the optimization, three PCR products in one tube had good specificity and average amplification efficiency. The concentration of primers was showed in Table 1, and then the result of agarose gel electrophoresis was shown in Figure 1.

Bottom Line: Multiplex PCR did not produce any non-specific amplicons when 149 related species or genera of standard bacteria were tested (100% specificity).Using recombinant plasmid and target pathogens, we were able to detect positive hybridization signals with 102 copies/muL and 103 cfu/mL per reaction.This assay was a specific and sensitive tool for simultaneous detection and identification of the new subtype of two pathogens causing diarrhea in human.

View Article: PubMed Central - HTML - PubMed

Affiliation: Beijing Institute of Radiation Medicine, Beijing, 100850, China. dazhijin@163.com

ABSTRACT

Background: The rapid and accurate detection and identification of the new subtype of the pathogens is crucial for diagnosis, treatment and control of the contagious disease outbreak. Here, in this study, an approach to detect and identify Escherichia coli O157:H7 and Vibrio cholerae O139 was established using oligonucleotide microarray. We coupled multiplex PCR with oligonucleotide microarray to construct an assay suitable for simultaneous identification of two subtypes of the pathogens.

Results: The stx1, stx2 gene and uidA gene having the specific mutant spot were chosen as the targets for Escherichia coli O157:H7, and meanwhile the ctxA, tcpA, and LPSgt gene for Vibrio cholerae O139. The oligonucleotide microarray was composed of eight probes including negative control and positive control from 16S rDNA gene. The six primers were designed to amplify target fragments in two triplex PCR, and then hybridized with oligonucleotide microarray. An internal control would be to run a PCR reaction in parallel. Multiplex PCR did not produce any non-specific amplicons when 149 related species or genera of standard bacteria were tested (100% specificity). In addition, Escherichia coli O157:H7 and Escherichia coli O157:non-H7, Vibrio cholerae O139 and Vibrio cholerae O1 had been discriminated respectively. Using recombinant plasmid and target pathogens, we were able to detect positive hybridization signals with 102 copies/muL and 103 cfu/mL per reaction.

Conclusion: The DNA microarray assay reported here could detect and identify Escherichia coli O157:H7 and Vibrio cholerae O139, and furthermore the subtype was distinguished. This assay was a specific and sensitive tool for simultaneous detection and identification of the new subtype of two pathogens causing diarrhea in human.

No MeSH data available.


Related in: MedlinePlus