Research article

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

Da-Zhi Jin^{* 1} , Xiao-Jing Xu^{* 1} , Su-Hong Chen¹ , Si-Yuan Wen¹ , Xue-En Ma³ , Zheng Zhang⁴ , Feng Lin² and Sheng-Qi Wang¹

¹Beijing Institute of Radiation Medicine, Beijing, 100850, China

²Wenzhou Medicine College affiliated Wenling First Hospital, Wenling, Zhejiang province, Wenling, 317500, China

³College of Animal Science and Medicine, Inner Mongolia Agricultural University, Hohhot, 010018, China

⁴Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, 310009, China

author email corresponding author email* Contributed equally

Infectious Agents and Cancer 2007, 2:23doi:10.1186/1750-9378-2-23

Published:	23 December 2007

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/μL 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.