实时荧光定量 RT-PCR 半反应优化检测 SARS-CoV-2。
RT-qPCR half-reaction optimization for the detection of SARS-CoV-2.
机构信息
Hospital de Clínicas de Porto Alegre, Centro de Pesquisa Experimental, Laboratório de Pesquisa em Resistência Bacteriana, Porto Alegre, RS, Brasil.
Hospital de Clínicas de Porto Alegre, Laboratório de Diagnóstico de SARS-CoV-2, Porto Alegre, RS, Brasil.
出版信息
Rev Soc Bras Med Trop. 2021 Dec 17;54:e03192020. doi: 10.1590/0037-8682-0319-2020. eCollection 2021.
INTRODUCTION
The main laboratory test for the diagnosis of coronavirus disease 2019 (COVID-19) is the reverse transcription real-time polymerase chain reaction (RT-qPCR). However, RT-qPCR is expensive because of the number of tests required. This study aimed to evaluate an alternative to the RT-qPCR approach for the detection of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is half of the total volume currently recommended by the US Centers for Disease Control and Prevention.
METHODS
The analytical limit of detection (LoD) and the reaction efficiency using half volumes of the RT-qPCR assay were evaluated for the N1 and N2 regions using a synthetic control RNA. A panel of 76 SARS-CoV-2-positive and 26 SARS-CoV-2-negative clinical samples was evaluated to establish clinical sensitivity and specificity.
RESULTS
The RT-qPCR assay efficiency was 105% for the half and standard reactions considering the N2 target and 84% (standard) and 101% (half) for N1. The RT-qPCR half-reaction LoD for N1 and N2 were 20 and 80 copies/µL, respectively. The clinical sensitivity and specificity were 100%. The half reaction presented a decrease of up to 5.5 cycle thresholds compared with standard RT-qPCR.
CONCLUSIONS
The use of the RT-qPCR half-reaction proved feasible and economic for the detection of SARS-CoV-2 RNA.
简介
新型冠状病毒病 2019（COVID-19）的主要实验室检测方法是逆转录实时聚合酶链反应（RT-qPCR）。然而，由于所需检测数量众多，RT-qPCR 成本较高。本研究旨在评估一种替代 RT-qPCR 方法，用于检测 SARS-CoV-2，其检测体积仅为美国疾病控制与预防中心目前推荐体积的一半。
方法
使用合成对照 RNA 评估 N1 和 N2 区域的 RT-qPCR 检测方法的分析检测限（LoD）和反应效率。评估了 76 份 SARS-CoV-2 阳性和 26 份 SARS-CoV-2 阴性临床样本，以确定临床灵敏度和特异性。
结果
考虑到 N2 靶标，对于 N1 和 N2，半反应和标准反应的 RT-qPCR 检测效率分别为 105%和 84%（标准）和 101%（半）。N1 和 N2 的 RT-qPCR 半反应 LoD 分别为 20 和 80 拷贝/µL。临床灵敏度和特异性均为 100%。与标准 RT-qPCR 相比，半反应降低了多达 5.5 个循环阈值。
结论
RT-qPCR 半反应的使用证明对于检测 SARS-CoV-2 RNA 是可行且经济的。
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