School of Food Engineering and Biotechnology, Hanshan Normal University, Chaozhou, China.
Department of Medical Laboratory, Huizhou Central People's Hospital, Huizhou, China.
Front Cell Infect Microbiol. 2021 Feb 1;11:613304. doi: 10.3389/fcimb.2021.613304. eCollection 2021.
BACKGROUND: The emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally. With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed. METHODS: Targeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed. For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively. For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA. For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively. RESULTS: The limit of detection was 1 copies/μl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens. Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples. CONCLUSION: This work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas.
背景:新型冠状病毒病 2019(COVID-19)的出现给全球公共卫生带来了挑战。随着实验室外对 SARS-CoV-2 检测需求的增加,迫切需要一种快速、准确的即时检测(POCT)方法。
方法:针对 SARS-CoV-2 的核衣壳(N)基因,设计了用于逆转录重组酶辅助扩增结合侧流层析试纸条(RT-RAA/LFD)平台的特异性引物和探针。为了评估特异性,分别用人冠状病毒、甲型流感病毒、乙型流感病毒、呼吸道合胞病毒和乙型肝炎病毒进行了测试。为了进行敏感性测定,使用 SARS-CoV-2 RNA 的重组质粒模板和假病毒进行了估计。为了进行临床评估,分别用定量逆转录 PCR(RT-qPCR)和 RT-RAA/LFD 对 100 份临床样本(SARS-CoV-2 阳性 13 份,阴性 87 份)进行了测试。
结果:在 RT-RAA/LFD 检测中,检测限为 1 拷贝/μl,可在 39°C 下 30 分钟内完成,与其他人类冠状病毒和临床呼吸道病原体无交叉反应。与 RT-qPCR 相比,该建立的 POCT 检测方法在检测临床样本时具有 100%的特异性和 100%的敏感性。
结论:本研究为 SARS-CoV-2 流行地区的疑似患者快速筛查、诊断和监测提供了一种便捷的 POCT 工具。
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