■耐碳青霉烯类肠杆菌(CRE)感染是对人类健康的紧迫威胁。本研究旨在开发和验证一种新型的多重实时PCR(多重qPCR)检测blaKPC,blaNDM,blaIMP,blaOXA-48-like,CRE分离株和临床样本中的blaVIM基因,以及将其与三种表型方法进行比较。
■评估了多qPCR测定的可靠性和检测限(LOD)。使用PCR和DNA测序作为鉴定CRE分离株和临床样品中碳青霉烯酶基因的参考方法。多重qPCR检测的准确性,改良碳青霉烯灭活法和EDTA改良碳青霉烯灭活法(mCIMandeCIM),基于碳青霉烯酶抑制剂的联合圆盘试验(CDT),并将基于胶体金的免疫层析试验与182株CRE的参考方法进行了比较。此外,收集112个临床样品以验证该多重qPCR测定的功效。
■多重qPCR测定的测定内和测定间的标准偏差(CV)为≤0.53%和≤2.04%,用于检测五种主要的碳青霉烯酶基因,分别;而LOD范围从2×102拷贝/mL到8×102拷贝/mL。PCR和DNA测序证实,182个CRE分离物中有168个产生碳青霉烯酶:KPC(n=93),NDM(n=46),IMP(n=8),OXA-48样(n=14),VIM(n=1),KPC&NDM(n=5),和KPC&NDM&IMP(n=1)。mCIMandeCIM的准确性,CDT,胶体金,多重qPCR检测率为96.2%,89.6%,100%,和100%分别用于检测碳青霉烯酶生产者。此外,多重qPCR检测的敏感性和特异性均为100%,用于检测临床样品中的每种碳青霉烯酶基因,与PCR和测序进行比较。
■对于临床分离株检测,多重qPCR检测与胶体金相当,优于mCIMandeCIM和CDT;而对于临床样本检测,它也显示出优异的性能。因此,多重qPCR检测在临床诊断中具有巨大的潜力。
UNASSIGNED: Carbapenem-resistant Enterobacterales (CRE) infection is an urgent threat to human health. This study aimed to develop and validate a novel multiplex real-time PCR (multi-qPCR) assay for the detection of the blaKPC, blaNDM, blaIMP, blaOXA-48-like, and blaVIM genes in CRE isolates and clinical samples, as well as to compare it with three phenotypic methods.
UNASSIGNED: The reliability and limit of detection (LOD) of the multi-qPCR assay were evaluated. PCR and DNA sequencing were used as the reference methods to identify carbapenemase genes in CRE isolates and clinical samples. The accuracy of the multi-qPCR assay, modified carbapenem inactivation and EDTA-modified carbapenem inactivation method (mCIMandeCIM), carbapenemase inhibitor-based combined disk test (CDT), and colloidal gold-based immunochromatographic test was compared with the reference methods with 182 isolates of CRE. Furthermore, 112 clinical samples were collected to validate the efficacy of this multi-qPCR assay.
UNASSIGNED: The standard deviations (CVs) of intra-assay and inter-assay of the multi-qPCR assay were ≤ 0.53% and ≤ 2.04% for detecting the five major carbapenemase genes, respectively; while the LOD ranged from 2×102 copies/mL to 8×102 copies/mL. PCR and DNA sequencing confirmed 168 out of 182 CRE isolates producing carbapenemase(s): KPC (n = 93), NDM (n = 46), IMP (n = 8), OXA-48-like (n = 14), VIM (n = 1), KPC&NDM (n = 5), and KPC&NDM&IMP (n = 1). The accuracy of mCIMandeCIM, CDT, Colloidal Gold, and the multi-qPCR assay was 96.2%, 89.6%, 100%, and 100% respectively for detecting carbapenemase(s) producers. Moreover, the sensitivity and specificity of the multi-qPCR assay were all 100% for the detection of each carbapenemase gene in clinical samples, compared with PCR and sequencing.
UNASSIGNED: For clinical isolate detection, the multi-qPCR assay is comparable to Colloidal Gold, and superior to mCIMandeCIM and CDT; while for clinical samples detection, it also shows excellent performance. Therefore, the multi-qPCR assay has great potential for clinical diagnosis.