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Published on: February 20, 2015
[Multiplex PCR assay for detection of virulence genes in avian pathogenic Escherichia coli]
Objective:
To efficiently study the virulence genes distribution of Avian pathogenic Escherichia coli (APEC), we developed four multiplex PCR to detect adhesin-associated genes, invasin and toxin-associated genes, serum resistance-associated genes and iron acquisition-associated genes.
Methods:
According to gene sequences published in GenBank, we designed and synthesized 18 specific primer pairs, which were used in the four multiplex PCR. Then, we determined the sensitivity of multiplex PCR using diluted bacterial or DNA templates. To verify the feasibility of these multiplex PCR, we determined the distribution of virulence genes in 100 APEC isolates using these multiplex PCR.
Results:
According to the results of PCR, we can conclude that each of the 18 genes was exactly and effectively amplified in the four multiplex PCR. The sensitivities of these four multiplex PCR were 10(3) Colony forming units (CFU), 10(3) CFU, 10(5) CFU, 10(5) CFU bacteria and 1ng, 1ng, 10ng and 10ng DNA, respectively. Furthermore, the results multiplex PCR for virulence genes distribution in 100 APEC were same as the single PCR.
Conclusion:
These results suggest that multiplex PCR developed in this study could efficiently detect the virulence genes of APEC, which was a useful and rapid technique for epidemiological investigation.
Insights
Four multiplex PCR assays were developed to efficiently detect virulence genes in Avian pathogenic Escherichia coli (APEC). These assays offer a rapid and effective method for epidemiological studies of APEC.
Area of Science:
- Veterinary Microbiology
- Molecular Biology
- Genetics
Background:
- Avian pathogenic Escherichia coli (APEC) causes significant economic losses in poultry industry.
- Understanding the distribution of virulence genes in APEC is crucial for effective disease control.
Purpose of the Study:
- To develop and validate multiplex PCR assays for efficient detection of APEC virulence genes.
- To determine the distribution of specific virulence-associated genes in APEC isolates.
Main Methods:
- Design and synthesis of 18 primer pairs for four multiplex PCR assays.
- Detection of adhesin, invasin, toxin, serum resistance, and iron acquisition genes.
- Sensitivity determination using bacterial and DNA templates.
- Validation in 100 APEC isolates.
Main Results:
- All 18 target genes were effectively amplified by the four multiplex PCR assays.
- High sensitivity achieved, with detection limits as low as 10^3 CFU/mL for bacteria and 1 ng for DNA.
- Multiplex PCR results for virulence gene distribution in 100 APEC isolates were consistent with single PCR.
Conclusions:
- The developed multiplex PCR assays provide an efficient, rapid, and reliable method for detecting APEC virulence genes.
- This technique is valuable for epidemiological investigations and understanding APEC pathogenicity.
