Droplet Digital PCR For Detection Of Fluoroquinolone-resistant Campylobacter Jejuni

Campylobacter jejuni is one of the main foodborne pathogens that cause human diarrhea in worldwide,with poultry being one of the main hosts of Campylobacter jejuni.Fluoroquinolone antibiotics are important drugs for the treatment of bacterial diarrhea,but with the use of antibiotics,the resistance rate of Campylobacter jejuni to fluoroquinolone antibiotics is increasing.Determining the resistance characteristics of pathogenic bacteria is important for antibiotic selection,but traditional methods rely on bacterial isolation and susceptibility testing,which is time-consuming and laborious,and it is difficult to quickly identify the occurrence of resistance mutations in highly abundant drug-resistant strains.Therefore,there is an urgent need for a rapid and precise detection technique for fluoroquinolone-resistant Campylobacter jejuni.Mutation of threonine(Thr)at GyrA 86 to isoleucine(Ile)is the main cause of Fluoroquinolone resistance in Campylobacter jejuni.Based on the key mutation sites of the gyrA gene,this study designed a pair of universal primers and three specific probes for the wild type(WT),mutant A256G(MT1),and mutant C257T(MT2)of the gyrA gene,and established a gyrA ddPCR detection technology based on point mutation recognition for the rapid detection of fluoroquinolone-resistant Campylobacter jejuni.Specific tests have shown that this method can specifically identify the wild and mutant types of Campylobacter jejuni gyrA and do not cross-react with other bacteria such as E.coli.Sensitivity tests showed that the detection limit of this method was less than 6 copies/reaction.This study further compares the resolution capabilities of Sanger sequencing,qPCR,and ddPCR for the detection of wild-type and mutant mixtures.The results showed that gyrA ddPCR could accurately detect gyrA mutant in a wildtype/mutant ratio of 1000:1,while the Sanger sequencing or qPCR method only accurately detected gyrA mutant at a wild-type/mutant ratio of 1:1 or 10:1.The results show that the gyrA ddPCR method can quickly identify gyrA resistant mutations in a high wild-type background compared with Sanger sequencing and qPCR,with better resolution ability.In this study,52 chicken samples were detected using the gyrA ddPCR method,of which 4 samples were mixed infections of Campylobacter jejuni WT and MT1,of which 1.7%,28.6%,53.3% and 87.0%were MT1,and 1 sample was a mixture of Campylobacter jejuni WT and MT2,with MT2 accounting for 66.4%.In addition,the gyrA ddPCR method was used to successfully monitor the process of drug-resistant mutations in the wild type of Campylobacter jejuni under the pressure of ciprofloxacin antibiotics,and the dominant subpopulation of mutations in the mutant type(C257T)was successfully monitored,further indicating that this method can be used for the rapid detection of Fluoroquinolones Campylobacter jejuni.Based on the Fluoroquinolone resistance mutation site of Campylobacter jejuni,this study established the gyrA ddPCR method,which has high sensitivity and specificity,especially in the context of high wild type,which can accurately identify low-abundance fluoroquinoloneresistant Campylobacter jejuni.It provides an effective means for the rapid clinical detection of fluoroquinolone-resistant Campylobacter jejuni and helps to clinically guide the correct use of fluoroquinolones.