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.
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.
Authors: Dossêh Jean Apôtre Afayibo; Hong Zhu; Beibei Zhang; Lan Yao; Hosny Ahmed Abdelgawad; Mingxing Tian; Jingjing Qi; Yali Liu; Shaohui Wang Journal: Vet Sci Date: 2022-06-25