To establish a universal touchdown PCR(TD-PCR)protocol to detect the major antibiotic resistance genes in Escherichia coli(E.coli),primers with varying melting temperatures(Tm)and various product and primer lengths based on the yeeJ gene of E.coli standard strain K12 MG1655,and twenty-nine primer pairs for the partial resistance genes of beta-lactamases,aminoglycosides,fluoroquinolones,carbapenems,and polymyxins,registered in GenBank,were designed and synthesized.Using the total DNA from E.coli standard strain K12 MG1655 as a template,the yeeJ gene was amplified by TD-PCR and conventional PCR protocols using primers P1 to P8 with different Tm values,which was to evaluate the specificity and applicability of primers with different Tm for TD-PCR amplification.Primers P9 to P16 were used to amplify the yeeJ gene with varying product lengths,and the specificity of the primers was assessed for TD-PCR amplification of products of different lengths.Primers P17 to P22 with different lengths were used to amplify the yeeJ gene,and the specificity of the primers was assessed for TD-PCR.Primer P5 was utilized to amplify the yeeJ gene,allowing for the analysis of the average brightness value of the target bands and the detection of the product yield in the TD-PCR protocol.Results of amplification using primers with different Tm indicated that,in comparison to the conventional PCR,the TD-PCR exhibited excellent specificity across all 8 primer pairs.The target bands were observed at the expected length,with no non-specific bands detected,suggesting that the TD-PCR had superior specificity and a broader range of applicable primer Tm values.TD-PCR showed better specificity than the conventional PCR protocol for all six pairs of primers with varying lengths,as evidenced by target bands appearing near the expected size with non-specific bands.Analysis of amplification product yield revealed that the average brightness value of target bands increased with the total number of reaction cycles in both TD-PCR and conventional PCR.However,the average brightness value was consistently higher in the target bands amplified by the conventional PCR,indicating a lower amplification product yield in the TD-PCR.The total DNA of 24 clinical isolates from geese was used as a template,and 29 pairs of resistance gene primers were utilized to amplify the partial resistance genes(beta-lactamases,aminoglycosides,fluoroquinolones,carbapenems,and polymyxins)by TD-PCR and conventional PCR,aiming at evaluating the effectiveness of TD-PCR in amplifying resistance genes.Clinical application results demonstrated a perfect match rate of 100％between the results obtained from amplifying the 29 pairs of resistance gene primers using the TD-PCR and conventional PCR protocols.The TD-PCR exhibited improved specificity compared to the conventional PCR protocol for amplifying various resistance gene primers,significantly reducing non-specific bands such as aac(3')-Ⅰa,aac(3')-Ⅱc,aac(3')-Ⅳ,and aac(6')-Ⅰb.These results demonstrate the successful establishment of a universal TD-PCR protocol with high specificity for detecting the major antibiotic resistance genes in E.coli,offering a convenient and efficient technical approach for studying bacterial resistance.
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