Development and evaluation of a dot blot assay for rapid determination of invasion-associated gene ibeA directly in fresh bacteria cultures of E. coli.
The ibeA gene, one of the important invasion-associated genes in neonatal meningitis Escherichia coli (NMEC), has been recently detected in avian pathogenic E. coli (APEC). Thus, it is necessary to close monitor the possible contamination of the poultry farms and its products to people. Here, a dot blot method for detecting the ibeA gene in E. coli was developed and validated. For the present study, probe sequence was designed and optimized for the specificity of dot blot. A 342-bp conserved fragment of ibeA gene was selected and labeled with digoxigenin (DIG)-dUTP according to the manufacturer's guidelines, which indicated that this probe hybridizes with ibeA. In our established method, the bacteria culture samples were directly spotted on the membrane, following simple lyses on the membrane. Hence, the extraction of genomic DNA is not required, which reduces the workload and shortens the time. Furthermore, this assay was very sensitive, which could detect as few as 2.5 × 10(3) CFU bacteria. The diagnostic reliability of this dot blot was evaluated on 467 APEC bacteria samples by using PCR analysis. Both methods showed that the result was in complete concordance. The dot blot assay was proved to be a simple, rapid, highly accurate, and cost-effective method to identify invasion-associated genes ibeA, which could be applied for initial screening of a large number of clinical samples or direct detection of bacteria culture.
Key Lab of Animal Bacteriology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, China. niuchunling02@163.com
Chunling
C
Niu
Shaohui
S
Wang
Chengping
C
Lu
D000818:Animals; D001431:Bacteriological Techniques / Q000191:economics / Q000379:methods*; D002353:Carrier State / Q000382:microbiology / Q000662:veterinary; D002645:Chickens; D004076:Digoxigenin / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000382:microbiology / Q000662:veterinary; D029968:Escherichia coli Proteins / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D037521:Virulence Factors / Q000235:genetics*
C096350:CusC protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D037521:Virulence Factors; D004076:Digoxigenin