Jiménez Karen Marcela, Fonseca-Mendoza Dora Janeth, Morel Adrien, Ortega-Recalde Oscar, Contreras Bravo Nora Constanza, Velandia-Piedrahita Camilo Andres, Steevens Adriana, Caldas Luisa Daniela, Ribón Juan Pablo, Sánchez Martha, Restrepo Carlos Martin, Cabrera Rodrigo
Laboratorio de Biología Molecular y Pruebas Diagnósticas de Alta Complejidad, Fundación Cardioinfantil-Instituto de Cardiología, Bogotá 110131, Colombia.
Center for Research in Genetics and Genomics-CIGGUR, GENIUROS Research Group, School of Medicine and Health Sciences, Universidad del Rosario, Bogotá 111221, Colombia.
Methods Protoc. 2022 Sep 21;5(5):73. doi: 10.3390/mps5050073.
RT-PCR tests have become the gold standard for detecting the SARS-CoV-2 virus in the context of the COVID-19 pandemic. Because of the extreme number of cases in periodic waves of infection, there is a severe financial and logistical strain on diagnostic laboratories. For this reason, alternative implementations and validations of academic protocols that employ the lowest cost and the most widely available equipment and reagents found in different regions are essential. In this study, we report an alternative implementation of the EUA 2019-nCoV CDC assay which uses a previously characterized duplex PCR reaction for the N1 and RNAse P target regions and an additional uniplex reaction for the N2 target region. Taking advantage of the Abbott m2000 Sample Preparation System and NEB Luna Universal Probe One-Step RT-qPCR kit, some of the most widely available and inexpensive nucleic acid extraction and amplification platforms, this modified test shows state-of-the-art analytical and clinical sensitivities and specificities when compared with the Seegene Allplex-SARS-CoV-2 assay. This implementation has the potential to be verified and implemented by diagnostic laboratories around the world to guarantee low-cost RT-PCR tests that can take advantage of widely available equipment and reagents.
在新冠疫情背景下，逆转录聚合酶链反应（RT-PCR）检测已成为检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）病毒的金标准。由于感染的周期性浪潮中病例数量极多，诊断实验室面临着严重的财务和后勤压力。因此，采用不同地区成本最低且最易获得的设备和试剂的学术方案的替代实施和验证至关重要。在本研究中，我们报告了美国食品药品监督管理局（FDA）紧急使用授权（EUA）的2019新型冠状病毒疾病预防控制中心（2019-nCoV CDC）检测法的一种替代实施方法，该方法使用先前已表征的针对N1和核糖核酸酶P（RNAse P）靶区域的双重聚合酶链反应（duplex PCR）以及针对N2靶区域的额外单重反应。利用雅培m2000样本制备系统和新英格兰生物实验室（NEB）Luna通用探针一步法逆转录定量聚合酶链反应（RT-qPCR）试剂盒，这两种最易获得且价格低廉的核酸提取和扩增平台，与Seegene Allplex-SARS-CoV-2检测法相比，这种改良检测法显示出了先进的分析和临床敏感性及特异性。这种实施方法有可能被世界各地的诊断实验室验证和采用，以确保能够利用广泛可用的设备和试剂进行低成本的RT-PCR检测。
