全基因组测序和real-time PCR法检测食源性沙门氏菌parC、gyrA基因突变特征

Characterization of parC and gyrA Mutations in Foodborne Salmonella Isolates by Whole-Genome Sequencing and Real-time PCR

以45株食源性沙门氏菌喹诺酮耐药株为对象,采取全基因组测序和实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)法检测parC、gyrA基因突变位点,并对检测方法可靠性、耐药基因突变特征进行评估和分析。首先将4株沙门氏菌进行二代全基因组测序,根据测序数据分析结果,建立了一种real-time PCR法检测gyrA Asp87Tyr、gyrA Asp87Asn、parC Thr57Ser和parC Ser80Ile这4个突变位点。将沙门氏菌进行qnrS、qnrA、qnrB的real-time PCR检测,发现有31株菌未检出qnr基因。以这31株菌为对象,采取real-time PCR法筛查基因突变位点,结果发现parC Thr57Ser和gyrA Asp87Asn型突变最常见。将real-time PCR阳性的10株菌扩增parC、gyrA基因全长并测序,real-time PCR检测和测序结果完全吻合,说明了real-time PCR检测的可靠性。全基因组测序和real-time PCR法相结合的方法用于耐药基因突变筛查,既可以发现新的基因突变,又可以快速筛查大样本的主要突变类型,可作为沙门氏菌耐药性研究的一种可靠手段。

A total of 45 quinolone-resistant foodborne Salmonella isolates were used to screen for mutations in the parC and gyrA genes by whole-genome sequencing(WGS)and real-time polymerase chain reaction(real-time PCR).The reliability of the detection methods was tested,and the mutation characteristics of the antibiotic-resistance genes were evaluated.Four strains were subjected to next-generation WGS,and according to the results obtained,a real-time PCR assay was developed to detect the mutation sites of Asp87Tyr and Asp87Asn in gyrA,and Thr57Ser and Ser80Ile in parC.The Salmonella isolates were subjected to PCR detection for the qnrS,qnrA and qnrB genes.Thirty-one strains which were not found to carry the qnr genes were screened for mutation sites by real-time PCR,revealing that mutations of parC Thr57Ser and gyrA Asp87Asn were found to be the most frequent mutations.In order to check the accuracy of real-time PCR,PCR amplification of the complete sequences of gyrA and parC was performed on 10 positive strains and the resulting amplicons were sequenced.The sequencing results were completely consistent with the real-time PCR results.The combination of WGS and real-time PCR,which can be used to detect new mutations in genes conferring quinolone resistance and rapidly screen for the main type of mutation in massive samples,is a rapid and reliable method for the identification of gyrA and parC mutations in Salmonella.