Gong Xinran, Yang Guang, Liu Wei, Wu Di, Duan Chunyuan, Jia Xinjing, Li Zhiqiang, Zou Xiaocang, Yu Renfeng, Zou Dayang, Wang Yong
School of Public Health, China Medical University, Shenyang, China.
Chinese PLA Center for Disease Control and Prevention, Beijing, China.
Front Microbiol. 2024 Mar 13;15:1279186. doi: 10.3389/fmicb.2024.1279186. eCollection 2024.
Recently, 10 plasmid-mediated mobile colistin resistance genes, to , and their variants have been identified, posing a new threat to the treatment of clinical infections caused by Gram-negative bacteria. Our objective was to develop a rapid, sensitive, and accurate molecular assay for detecting genes in clinical isolates.
The primers and corresponding TaqMan-MGB probes were designed based on the sequence characteristics of all reported MCR family genes, multiplex Taqman-MGB probe-based qPCR assays were developed and optimized, and the sensitivity, specificity and reproducibility of the method were evaluated. The assay contained 8 sets of primers and probes in 4 reaction tubes, each containing 2 sets of primers and probes.
The standard curves for both the single and multiplex systems showed good linearity (R > 0.99) between the starting template amount and the Ct value, with a lower limit of detection of 10 copies/μL. The specificity test showed positive amplification results only for strains containing the genes, whereas the other strains were negative. The results of intra-and inter-group repeatability experiments demonstrated the stability and reliability of the newly developed method. It was used to detect genes in 467 clinically-obtained Gram-negative isolates, which were multidrug-resistant. Twelve strains containing the genes were detected (seven isolates carrying , four isolates carrying , and one isolate carrying ). The products amplified by the full-length PCR primer were identified by sequencing, and the results were consistent with those of the multiplex qPCR method.
The assay developed in this study has the advantages of high specificity, sensitivity, and reproducibility. It can be used to specifically detect drug-resistant clinical isolates carrying the genes (), thus providing a better basis for clinical drug treatment and drug resistance research.
最近，已鉴定出10种质粒介导的可移动黏菌素耐药基因及其变体，这对革兰氏阴性菌引起的临床感染治疗构成了新威胁。我们的目的是开发一种快速、灵敏且准确的分子检测方法，用于检测临床分离株中的这些基因。
根据所有已报道的MCR家族基因的序列特征设计引物和相应的TaqMan-MGB探针，开发并优化基于多重TaqMan-MGB探针的qPCR检测方法，并评估该方法的灵敏度、特异性和可重复性。该检测方法在4个反应管中包含8组引物和探针，每个反应管包含2组引物和探针。
单重和多重系统的标准曲线在起始模板量与Ct值之间均显示出良好的线性关系（R＞0.99），检测下限为10拷贝/μL。特异性测试显示，仅含有这些基因的菌株呈现阳性扩增结果，而其他菌株为阴性。组内和组间重复性实验结果证明了新开发方法的稳定性和可靠性。用该方法检测了467株临床获得的多重耐药革兰氏阴性分离株中的这些基因。检测到12株含有这些基因的菌株（7株携带mcr-1，4株携带mcr-2，1株携带mcr-3）。对全长PCR引物扩增的产物进行测序鉴定，结果与多重qPCR方法一致。
本研究开发的检测方法具有高特异性、灵敏度和可重复性的优点。它可用于特异性检测携带这些基因（mcr）的耐药临床分离株，并为临床药物治疗和耐药性研究提供更好的依据。
