Limits...
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

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

License
getmorefigures.php?uid=PMC2267443&req=5

Mentions: The 60 candidate probes were spotted in an array format of 6 rows × 10 columns. The PCR products labeled by fluorescence were mixed with hybridization solution and then hybridized with the probe array. The suitable probe for each gene was selected according to the specificity and hybridization signal intensity, which was analyzed by using GenePix 4.0 software program. The screening result of candidate probes was shown in Figure 2, and then the hybridization signal intensity corresponding to each candidate probe was shown in Figure 3. The statistical analysis of the quantified signal intensities indicated: stx1 No.7 probe, stx2 No.10 probe, uidA No.9 probe, and LPSgt No. 10 probe, had stronger signal intensity than that of other probes, at any same level of the PCR template amount, and in the meantime there are no non-specific signals. No.9 probe of uidA was a shorter probe (19 bp) containing +93 mutant spot in the middle of the sequence. With comparison to the No. 10 probe (without mutant point), this probe was strictly specific to the single base mutant uidA gene of E. coli O157:H7, indicating its applicability in detection of the uidA gene mutation spot. No.3, No.4, and No.6 probes for ctxA had statistically proximal signal intensities. Nevertheless, No.4 probe was more specific than the others. No.2, No.5, and No.9 probes for tcpA also had statistically proximal signal intensities at higher level of DNA template amount; whereas, No.9 probe had apparently higher signal intensities than No.2, No.5 probes at lower level of DNA template amount.


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 60 candidate probes were spotted in an array format of 6 rows × 10 columns. The PCR products labeled by fluorescence were mixed with hybridization solution and then hybridized with the probe array. The suitable probe for each gene was selected according to the specificity and hybridization signal intensity, which was analyzed by using GenePix 4.0 software program. The screening result of candidate probes was shown in Figure 2, and then the hybridization signal intensity corresponding to each candidate probe was shown in Figure 3. The statistical analysis of the quantified signal intensities indicated: stx1 No.7 probe, stx2 No.10 probe, uidA No.9 probe, and LPSgt No. 10 probe, had stronger signal intensity than that of other probes, at any same level of the PCR template amount, and in the meantime there are no non-specific signals. No.9 probe of uidA was a shorter probe (19 bp) containing +93 mutant spot in the middle of the sequence. With comparison to the No. 10 probe (without mutant point), this probe was strictly specific to the single base mutant uidA gene of E. coli O157:H7, indicating its applicability in detection of the uidA gene mutation spot. No.3, No.4, and No.6 probes for ctxA had statistically proximal signal intensities. Nevertheless, No.4 probe was more specific than the others. No.2, No.5, and No.9 probes for tcpA also had statistically proximal signal intensities at higher level of DNA template amount; whereas, No.9 probe had apparently higher signal intensities than No.2, No.5 probes at lower level of DNA template amount.

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