Establishment of PCR detection methods for drug-resistance gene mcr-8

Abstract

The emergence of bacterial resistance seriously threatens the sustainable development of public health and animal husbandry. Polymyxin is a key drug for clinical treatment of gram-negative bacteria infection and the last line of defense to prevent and control bacterial infection. The mobile colistin resistance-8 (mcr-8) gene is one of the main genes that cause bacteria to develop polymyxin resistance. This study designed and established a specific PCR detection method for the mcr-8 gene to be used to quickly screen and monitor the mcr-8 gene. According to the mcr-8 gene sequence, a pair of specific primers were designed using the primer design software Oligo 7 to amplify the conserved region of the target gene mcr-8.1 (101-1798), and the theoretical amplification product length was 1268 bp. The physical and chemical properties of the designed primers meet the requirements, their GC content is moderate (47.6% and 52.6%), and the Tm value is stable at 59.4℃. Specific verification showed that samples containing only the mcr-8 gene showed 1268 bp specific amplification products, and no target bands were observed in the negative control group; the positive control PCR product was sequenced, and the BLAST comparison showed that the sequence consistency with the mcr-8 gene was 100%, which confirmed that the method was highly specific. Sensitivity analysis shows that the lower limit of detection of mcr-8 plasmid DNA in this system can reach 10 pg. Through detection of 15 clinical isolates (including 10 E. coli and 5 Salmonella strains), the mcr-8 gene was not detected in all the tested strains. The PCR detection method of mcr-8 gene established in this study provides effective technical means for the rapid detection of bacterial resistance, and has important application value in the fields of clinical treatment and bacterial resistance monitoring.