利用环介导等温扩增（LAMP）技术快速检测新型 B1-β-内酰胺酶基因 blaAFM-1。
Rapid detection of a novel B1-β-lactamase gene, blaAFM-1 using a loop-mediated isothermal amplification (LAMP) assay.
机构信息
Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
出版信息
Ann Clin Microbiol Antimicrob. 2021 Dec 7;20(1):80. doi: 10.1186/s12941-021-00486-z.
BACKGROUND
BlaAFM-1 (GenBank Accession No. 143105.1) is a new B1 subclass metallo-β-lactamase gene discovered by our group, and isolated from an Alcaligenes faecalis plasmid that renders carbapenem antibiotics ineffective. In this study, we generated a fast and reliable assay for blaAFM-1 detection.
METHODS
We designed optimum loop-mediated isothermal amplification (LAMP) primers and constructed a recombinant plasmid AFM-1 to specifically detect blaAFM-1. Optimal LAMP primers were used to assess sensitivity of the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (simulated sputum and simulated feces). Fifty two samples, without blaAFM-1, were used to assess LAMP real-time assay specificity; these samples were verified by conventional PCR and sequencing for the absence of blaAFM-1. Three hundred clinical Gram-negative carbapenem-resistant strains were tested by LAMP assay for strains carrying blaAFM-1, which were confirmed by conventional PCR and Sanger sequencing. We calculated the sensitivity and its 95% confidence interval (95% CI), specificity and its 95% CI, and predictive values of the LAMP assay and conventional PCR/sequencing by investigating positive and negative clinical strains.
RESULTS
The lowest limit of detection for the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (in both simulated sputum and simulated feces) was 10 copies/reaction. All amplification curves of the 52 blaAFM-1-free bacteria strains were negative, suggesting the LAMP assay had excellent specificity for detecting blaAFM-1. Among the 300 clinical strains, eight were positive for blaAFM-1 using LAMP. These LAMP results were consistent with conventional PCR and Sanger sequencing data. As with conventional PCR/sequencing, the LAMP method exhibits 100% sensitivity (95% CI 59.8-100%) and 100% specificity (95% CI 98.4-100%) for blaAFM-1 detection. The LAMP assay is also time-efficient (1 h) for blaAFM-1 detection.
CONCLUSIONS
We established a new LAMP assay with high sensitivity and specificity to detect the novel B1-β-lactamase gene, blaAFM-1.
背景
BlaAFM-1（GenBank 登录号：143105.1）是我们小组发现的一种新的 B1 亚类金属β-内酰胺酶基因，从一株使碳青霉烯类抗生素失效的粪产碱杆菌质粒中分离得到。本研究旨在建立一种快速可靠的 blaAFM-1 检测方法。
方法
我们设计了最佳的环介导等温扩增（LAMP）引物，并构建了 AFM-1 重组质粒，用于特异性检测 blaAFM-1。使用最佳的 LAMP 引物评估了重组质粒 AFM-1 和 blaAFM-1 补充样本（模拟痰和模拟粪便）的敏感性。52 份无 blaAFM-1 的样本用于评估 LAMP 实时检测的特异性；这些样本通过常规 PCR 和测序来验证无 blaAFM-1。300 株临床革兰氏阴性碳青霉烯类耐药菌株通过 LAMP 检测携带 blaAFM-1 的菌株，通过常规 PCR 和 Sanger 测序进行验证。通过调查阳性和阴性临床菌株，计算了 LAMP 检测和常规 PCR/测序的敏感性及其 95%置信区间（95%CI）、特异性及其 95%CI 和预测值。
结果
重组质粒 AFM-1 和 blaAFM-1 补充样本（模拟痰和模拟粪便）的最低检测限为 10 个拷贝/反应。52 株无 blaAFM-1 的细菌的所有扩增曲线均为阴性，表明 LAMP 检测法对 blaAFM-1 的检测具有良好的特异性。在 300 株临床菌株中，有 8 株通过 LAMP 检测到 blaAFM-1 呈阳性。这些 LAMP 结果与常规 PCR 和 Sanger 测序数据一致。与常规 PCR/测序一样，LAMP 法对 blaAFM-1 的检测具有 100%的敏感性（95%CI 59.8-100%）和 100%的特异性（95%CI 98.4-100%）。LAMP 检测法检测 blaAFM-1 的时间也很高效（1 小时）。
结论
我们建立了一种新的 LAMP 检测法，具有高灵敏度和特异性，可用于检测新型 B1-β-内酰胺酶基因 blaAFM-1。
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