Development and evaluation of a dot blot assay for rapid determination of invasion-associated gene ibeA directly in fresh bacteria cultures of E. coli

Abstract

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.

Specific Properties of Enteropathogenic Escherichia coli Isolates from Diarrheal Patients and Comparison to Strains from Foods and Fecal Specimens from Cattle, Swine, and Healthy Carriers in Osaka City, Japan

“…Group I strains were further divided into subgroups Ia and Ib depending on whether they contained the gene with the strongest association with diarrhea, efa1 (lifA). The 14 pairs of primers (including three variants of lpfA) and the PCR conditions used in this study are listed in Table 1 (15)(16)(17)(20)(21)(22)(23)(24).…”

Design of dot-blot hybridization assay for simultaneous detection of Campylobacter jejuni and Campylobacter coli: a preliminary study

“…In a study by Niu and colleagues in 2012, PCR and dot-blot techniques were used to detect the ibeA gene in E. coli . Their results showed that dot-blot assay is a rapid and highly accurate method for screening clinical samples 34 . In Sun’s study in 2005, variable regions of the 16S gene of E. coli , P. mirabilis , K. oxytoca , and P. aeruginosa were selected for designing oligonucleotide probes in a sensor assay.…”

Deletion of genes yadC and yicS from avian pathogenic Escherichia col (APEC) strain SCI-07 and their role in pathogenicity

“…The infection of birds may occur via the eggshell, navel of newborn chicks and digestive tract, due the contact with contaminated feaces and the inhalation of contaminated dust. Apart from the control of environmental conditions such as humidity, ventilation and chlorination of drinking water, prevention of APEC infection usually relies on antibiotic therapy (Niu et al, 2012). The extensive use of antibiotics created an increase in the level of avian E. coli resistance to antibiotics (Zhao et al, 2005), which can be transferred to Human…”

“…Extraintestinal pathogenic E. coli (ExPEC) strains. The possibility about the rising of multidrug resistance strains which potently can reach human beings claims from public health policies against the use of antibiotics in food animal production (Niu et al, 2012), and creates an urgent need of vaccine strains presenting common antigenic epitopes to several APEC serogroups.…”