Federal State Budgetary Institution Centre for Strategic Planning and Management of Biomedical Health Risks of the Federal Medical Biological Agency, Moscow, Russian Federation.
G.N. Speranskiy Children Hospital No. 9, Moscow, Russian Federation.
Microbiol Spectr. 2022 Aug 31;10(4):e0051622. doi: 10.1128/spectrum.00516-22. Epub 2022 Jul 11.
Loop-mediated isothermal amplification (LAMP) is an alternative to PCR that is faster and requires fewer resources. Here, we describe two LAMP assays for the detection of human adenoviruses in the feces of children with acute intestinal infections. We designed сolorimetric LAMP (c-LAMP) and real-time LAMP (f-LAMP) with fluorescent probes to detect the DNA of the adenovirus F human adenovirus 40/41 (hAdV40/41) hexon gene. The detection limit of both developed methods was 10 copies/mL, which is comparable to the sensitivity of PCR. The specificities of both c-LAMP and f-LAMP were high, with no false-positive results for clinical samples that do not contain adenovirus F, when testing other viruses and microorganisms. Comparative tests of PCR and LAMP on clinical samples from patients with acute gastroenteritis were carried out. For all samples with a PCR threshold cycle () of up to 36, the PCR and LAMP results completely coincided; however, at low viral loads, the diagnostic sensitivity of LAMP, especially c-LAMP with colorimetric detection, was inferior to that of PCR. The combination of LAMP with modern methods of nucleic acid extraction, both in manual and automatic modes, can reduce the time for a complete study, including extraction of nucleic acid material and amplification, to 60 min. In April 2022, several cases of acute hepatitis of unknown origin were reported in children from 12 countries. In many cases, enteric adenovirus or SARS-CoV-2 and adenovirus coinfection were detected. It is known that human adenoviruses can cause different infections of varying severity, from asymptomatic to severe cases with lethal outcomes. There is a need to increase the diagnostic capabilities of clinical laboratories to identify such an underestimated pathogen as adenovirus. Although PCR remains the gold standard for pathogen detection, this method requires specialized equipment and has a long turnaround time to process samples. Previously, LAMP assays for the detection of human adenovirus have been based on measuring the turbidity, the fluorescence of intercalated dyes, or electrophoretic separation. Herein, we present LAMP-based assays with colorimetric or fluorescent detection and perform a detailed assessment of their sensitivity, specificity, and diagnostic performance.
环介导等温扩增（LAMP）是一种替代 PCR 的方法，它更快且需要更少的资源。在这里，我们描述了两种用于检测急性肠道感染儿童粪便中人腺病毒的 LAMP 检测方法。我们设计了比色 LAMP（c-LAMP）和实时 LAMP（f-LAMP），使用荧光探针来检测腺病毒 F 人类腺病毒 40/41（hAdV40/41）六邻体基因的 DNA。两种方法的检测限均为 10 拷贝/mL，与 PCR 的灵敏度相当。当测试其他病毒和微生物时，c-LAMP 和 f-LAMP 的特异性都很高，对于不包含腺病毒 F 的临床样本没有假阳性结果。对来自急性胃肠炎患者的临床样本进行了 PCR 和 LAMP 的比较测试。对于所有 PCR 阈值循环（）高达 36 的样本，PCR 和 LAMP 的结果完全一致；然而，在低病毒载量下，LAMP 的诊断灵敏度，特别是具有比色检测的 c-LAMP，不如 PCR。LAMP 与现代核酸提取方法的结合，无论是手动还是自动模式，都可以将完整研究的时间，包括核酸材料的提取和扩增，缩短至 60 分钟。2022 年 4 月，报告了来自 12 个国家的儿童不明原因急性肝炎的几例病例。在许多情况下，检测到肠腺病毒或 SARS-CoV-2 和腺病毒合并感染。已知人类腺病毒可引起不同严重程度的感染，从无症状到致命的严重病例。需要提高临床实验室的诊断能力以识别这种被低估的病原体，如腺病毒。尽管 PCR 仍然是病原体检测的金标准，但该方法需要专门的设备，并且处理样本的周转时间较长。以前，用于检测人类腺病毒的 LAMP 检测方法基于测量浊度、嵌入染料的荧光或电泳分离。在此，我们提出了基于 LAMP 的比色或荧光检测方法，并对其灵敏度、特异性和诊断性能进行了详细评估。
