SoRelle Jeffrey A, Frame Ithiel, Falcon Alejandra, Jacob Jerin, Wagenfuehr Jennifer, Mitui Midori, Park Jason Y, Filkins Laura
Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX.
Department of Pathology and Laboratory Medicine, Children's Health System of Texas, Dallas, TX.
J Appl Lab Med. 2020 Sep 1;5(5):889-896. doi: 10.1093/jalm/jfaa089.
Detection of SARS-CoV-2 viral RNA is important for the diagnosis and management of COVID-19.
We present a clinical validation of a reverse transcription PCR (RT-PCR) assay for the SARS-CoV-2 nucleocapsid (N1) gene. Off-board lysis on an automated nucleic acid extraction system was optimized with endemic coronaviruses (OC43 and NL63). Genomic RNA and SARS-CoV-2 RNA in a recombinant viral protein coat were used as control materials and compared for recovery from nucleic acid extraction.
Nucleic acid extraction showed decreased recovery of endemic Coronavirus in vitro transcribed RNA (NL63) compared with attenuated virus (OC43). SARS-CoV-2 RNA had more reliable recovery from extraction through amplification than genomic RNA. Recovery of genomic RNA was improved by combining lysis buffer with clinical matrix before adding RNA. The RT-PCR assay demonstrated 100% in silico sensitivity and specificity. The accuracy across samples was 100% (75 of 75). Precision studies showed 100% intra-run, inter-run, and inter-technologist concordance. The limit of detection was 264 copies per milliliter (estimated 5 copies per reaction; 35.56 mean threshold cycle value).
This SARS-CoV-2 assay demonstrates appropriate characteristics for use under an Emergency Use Authorization. Endemic coronavirus controls were useful in optimizing the extraction procedure. In the absence of live or attenuated virus, recombinant virus in a protein coat is an appropriate control specimen type for assay validation during a pandemic.
检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）病毒RNA对于2019冠状病毒病（COVID-19）的诊断和管理至关重要。
我们展示了一种针对SARS-CoV-2核衣壳（N1）基因的逆转录聚合酶链反应（RT-PCR）检测方法的临床验证。使用地方性冠状病毒（OC43和NL63）对自动核酸提取系统上的离线裂解进行了优化。将重组病毒蛋白衣壳中的基因组RNA和SARS-CoV-2 RNA用作对照材料，并比较核酸提取后的回收率。
与减毒病毒（OC43）相比，核酸提取显示地方性冠状病毒体外转录RNA（NL63）的回收率降低。通过扩增，SARS-CoV-2 RNA从提取中获得的回收率比基因组RNA更可靠。在添加RNA之前，将裂解缓冲液与临床基质混合可提高基因组RNA的回收率。RT-PCR检测方法在计算机模拟中显示出100%的灵敏度和特异性。所有样本的准确率为100%（75个样本中的75个）。精密度研究显示批内、批间和技术人员间的一致性均为100%。检测限为每毫升264个拷贝（估计每个反应5个拷贝；平均阈值循环值为35.56）。
这种SARS-CoV-2检测方法在紧急使用授权下显示出合适的特性。地方性冠状病毒对照有助于优化提取程序。在没有活病毒或减毒病毒的情况下，蛋白衣壳中的重组病毒是大流行期间检测方法验证的合适对照样本类型。
