Université de Sétif 1, Faculté de Médecine, Centre Hospitalier Universitaire (CHU) de Sétif, Laboratoire de Microbiologie, Sétif, Algeria; Département des Sciences Naturelles, École Normale Supérieure Assia DJEBAR, Constantine, Algeria; Aix-Marseille Univ., IRD, APHM, MEPHI, IHU-Mediterranee Infection, 19-21 boulevard Jean Moulin, 13005 Marseille, France.
Université de Sétif 1, Faculté de Médecine, Centre Hospitalier Universitaire (CHU) de Sétif, Laboratoire de Microbiologie, Sétif, Algeria.
J Glob Antimicrob Resist. 2020 Mar;20:266-271. doi: 10.1016/j.jgar.2019.08.018. Epub 2019 Aug 30.
We aimed to develop here a specific real-time PCR assay with TaqMan® probe to detect efficiently bacterial strains harboring the new plasmid mediated-colistin resistance mcr-8 gene.
Specific primers and probe for mcr-8 gene were designed from sequences alignment of all mcr genes variants. Specificity of the designed primers and probe were first checked par BlastN analysis and by in silico PCR. The analytical sensitivity and specificity tests were performed in vitro on a panel of 290 genomic DNA of Gram-negative bacteria and 250 metagenomic DNA from human stool samples. Whole genome sequencing (WGS) was performed here using MiSeq technology.
Designed primers and probe were 100% specific tomcr-8 gene by BlastN and in silico PCR analysis. Real-time PCR screening of a collection of clinical isolates resulted to one positive Klebsiella pneumoniae isolate (KP95). WGS confirmed that this isolate harbored the mcr-8 gene and other resistance genes such as bla, bla β-lactamases. Our real-time PCR was highly sensitive on a 10-fold dilution serie from a calibrated inoculum at 10 CFU/mL with a limit of detection at 55 CFU/mL.
To the best of our knowledge, we propose here, the first real-time PCR assay targeting mcr-8 gene with high specificity and sensitivity, able to detect mcr-8 gene in less than 2 h from any DNA sample. This real-time PCR assay allowed the first description of a clinical K. pneumoniae strain harboring the mcr-8 gene in Algeria.
我们旨在开发一种新的 TaqMan®探针实时 PCR 检测方法，以有效检测携带新型质粒介导的粘菌素耐药基因 mcr-8 的细菌株。
从所有 mcr 基因变异体的序列比对中设计 mcr-8 基因的特异性引物和探针。首先通过 BlastN 分析和计算机 PCR 分析检查设计引物和探针的特异性。在体外对 290 株革兰氏阴性菌基因组 DNA 和 250 株人类粪便样本宏基因组 DNA 进行了分析灵敏度和特异性测试。使用 MiSeq 技术进行全基因组测序（WGS）。
通过 BlastN 和计算机 PCR 分析，设计的引物和探针对 mcr-8 基因具有 100%的特异性。对临床分离株的实时 PCR 筛选结果为一株阳性肺炎克雷伯菌（KP95）。WGS 证实该分离株携带 mcr-8 基因和其他耐药基因，如 bla、blaβ-内酰胺酶。我们的实时 PCR 对 10 倍稀释系列的校准接种物具有高度敏感性，在 10 CFU/mL 时检测限为 55 CFU/mL。
据我们所知，我们首次提出了一种针对 mcr-8 基因的高特异性和高灵敏度的实时 PCR 检测方法，能够在不到 2 小时内从任何 DNA 样本中检测到 mcr-8 基因。该实时 PCR 检测方法首次描述了在阿尔及利亚携带 mcr-8 基因的临床肺炎克雷伯菌菌株。
