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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.


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Mentions: The recombinant plasmids were quantified and prepared in 10-fold serial dilutions, which were used as the PCR templates to test the sensitivity of the oligonucleotide microarray. In the triplex PCR mixture, when the template plasmids were 103copies/μL, the PCR products can be detected both by using agarose gel electrophoresis and from the positive signal confirmed according to the cutoff value when hybridized with the detecting probe array; when the template plasmids were 102copies/μL, the PCR products, which can be detected only by hybridizing with the detecting probe array (the ratio of signal to noise was greater than 4), produce no visible bands on the same agarose gel electrophoresis. Furthermore, when the template plasmids were 10 copies/μL, PCR products can not be detected either by using agarose gel electrophoresis or by hybridization with the detecting probe array. The above results and the value of signal intensity corresponding to serial dilution were shown in Figure 6 and in Figure 7 respectively. Furthermore, the test limit of real time PCR was 102 copies/μL(data not shown). Similarly when serial dilution (10-fold) of two targets from 106~101cfu/mL were evaluated, the positive signals were observed with a detection limit of 103cfu/mL. The result of statistical analysis was shown in Figure 8. Meanwhile, the test limit of real time PCR and conventional microbiological methods was 103cfu/mL and 102cfu/mL respectively (data not shown).


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 recombinant plasmids were quantified and prepared in 10-fold serial dilutions, which were used as the PCR templates to test the sensitivity of the oligonucleotide microarray. In the triplex PCR mixture, when the template plasmids were 103copies/μL, the PCR products can be detected both by using agarose gel electrophoresis and from the positive signal confirmed according to the cutoff value when hybridized with the detecting probe array; when the template plasmids were 102copies/μL, the PCR products, which can be detected only by hybridizing with the detecting probe array (the ratio of signal to noise was greater than 4), produce no visible bands on the same agarose gel electrophoresis. Furthermore, when the template plasmids were 10 copies/μL, PCR products can not be detected either by using agarose gel electrophoresis or by hybridization with the detecting probe array. The above results and the value of signal intensity corresponding to serial dilution were shown in Figure 6 and in Figure 7 respectively. Furthermore, the test limit of real time PCR was 102 copies/μL(data not shown). Similarly when serial dilution (10-fold) of two targets from 106~101cfu/mL were evaluated, the positive signals were observed with a detection limit of 103cfu/mL. The result of statistical analysis was shown in Figure 8. Meanwhile, the test limit of real time PCR and conventional microbiological methods was 103cfu/mL and 102cfu/mL respectively (data not shown).

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