Cheng Jinzhi, Wang Yu, Zhou Yuhong, Lu Jingrun, Tang Xiaomin
School of Basic Medical Sciences, Guizhou Medical University, Guiyang, China.
Department of Clinical Laboratory, The First People's Hospital of Guiyang, Guiyang, Guizhou, China.
Front Microbiol. 2023 Mar 8;14:1121930. doi: 10.3389/fmicb.2023.1121930. eCollection 2023.
One of the main pathogens responsible for human hand, foot, and mouth disease (HFMD), coxsackievirus A16, has put young children's health at danger, especially in countries in the Asia-Pacific region. Early quick identification is essential for the avoidance and control of the disorder since there are no vaccinations or antiviral medications available to prevent and manage CVA16 infection.
Here, we describe the creation of an easy, speedy, and accurate CVA16 infection detection approach using lateral flow biosensors (LFB) and reverse transcriptionmultiple cross displacement amplification (RT-MCDA). A group of 10 primers was developed for the RT-MCDA system in order to amplify the genes in an isothermal amplification device while targeting the highly conserved region of the CVA16 VP1 gene. Then, without requiring any extra tools, RT-MCDA amplification reaction products might well be detected by visual detection reagent (VDR) and LFB.
The outcomes showed that 64°C within 40 min was the ideal reaction setting for the CVA16-MCDA test. Target sequences with <40 copies might be found using the CVA16-MCDA. There was no cross-reaction among CVA16 strains and other strains. The findings demonstrated that the CVA16-MCDA test could promptly and successfully identify all of the CVA16-positive (46/220) samples identified by the traditional real-time quantitative polymerase chain reaction (qRT-PCR) assays for 220 clinical anal swab samples. The whole process, such as the processing of the sample (15 min), the MCDA reaction (40 min), and the documenting of the results (2 min), could be finished in 1 h.
The CVA16-MCDA-LFB assay, which targeted the VP1 gene, was an efficient, simple, and highly specific examination that might be used extensively in rural regions' basic healthcare institutions and point-of-care settings.
人类手足口病（HFMD）的主要病原体之一柯萨奇病毒A16（CVA16），对幼儿健康构成威胁，尤其是在亚太地区国家。由于目前尚无预防和管理CVA16感染的疫苗或抗病毒药物，早期快速识别对于避免和控制该疾病至关重要。
在此，我们描述了一种使用侧向流动生物传感器（LFB）和逆转录多重交叉置换扩增（RT-MCDA）创建简单、快速且准确的CVA16感染检测方法。为RT-MCDA系统设计了一组10条引物，以便在等温扩增装置中扩增基因，同时靶向CVA16 VP1基因的高度保守区域。然后，无需任何额外工具，RT-MCDA扩增反应产物可通过视觉检测试剂（VDR）和LFB进行检测。
结果表明，40分钟内在64°C是CVA16-MCDA检测的理想反应条件。使用CVA16-MCDA可检测到少于40个拷贝的靶序列。CVA16菌株与其他菌株之间无交叉反应。结果表明，对于220份临床肛门拭子样本，CVA16-MCDA检测能够迅速且成功地识别出所有经传统实时定量聚合酶链反应（qRT-PCR）检测为CVA16阳性（46/220）的样本。整个过程，如样本处理（15分钟）、MCDA反应（40分钟）和结果记录（2分钟），可在1小时内完成。
靶向VP1基因的CVA16-MCDA-LFB检测是一种高效、简单且高度特异的检测方法，可广泛应用于农村地区的基层医疗机构和即时检测场所。
