用于 COVID-19 恢复期血浆供者和患者血液高通量检测的 SARS-CoV-2 RNA RT-PCR 检测方法的验证。

Validation of a SARS-CoV-2 RNA RT-PCR assay for high-throughput testing in blood of COVID-19 convalescent plasma donors and patients.

机构信息

Department of Transfusion Medicine and Hemostaseology, University Hospital of Erlangen, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany.

Institute of Clinical and Molecular Virology, University Hospital of Erlangen, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany.

出版信息

Transfusion. 2021 Feb;61(2):368-374. doi: 10.1111/trf.16178. Epub 2020 Nov 9.

DOI:10.1111/trf.16178

PMID:33125162

Abstract

BACKGROUND

The frequency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNAemia in blood donors is uncertain. Thus, assays for SARS-CoV-2 RNA detection in blood, validated on commercially available polymerase chain reaction (PCR) systems, are required to allow a good comparability of data.

STUDY DESIGN AND METHODS

The cobas SARS-CoV-2 dual-target reverse transcriptase PCR (RT-PCR) assay, licensed for respiratory swab SARS-CoV-2 RNA testing, was validated for detection of viral RNA in blood. For the validation panel, SARS-CoV-2-positive plasma samples were prepared by spiking SARS-CoV-2-positive respiratory specimens in negative human plasma. The 95% limit of detection (LOD95) was determined by probit analysis. For clinical validation, coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) donors and patients with COVID-19 with a severe disease course treated in an intensive care unit (ICU) were included.

RESULTS

The validation of the SARS-CoV-2 RT-PCR assay for blood demonstrated high sensitivity and specificity and intra- and inter-assay precision and efficiency. The LOD95 for SARS-CoV-2 RNA was 5.0 genome copies/mL (95% confidence interval [CI], 3.3-12 copies/mL) for target 1 and 4.3 genome copies/mL (95% CI, 2.9-10 copies/mL) for target 2. In a cohort of 39 CCP donors with 66 CCP donations no SARS-CoV-2 RNA in plasma was detected. Screening of 25 blood samples of 19 ICU patients with COVID-19 showed six positive results for SARS-CoV-2 RNA in at least one target of the assay.

CONCLUSION

The SARS-CoV-2 RNA assay, only licensed for respiratory swabs, performed on a PCR system for high-throughput testing, showed a good assay performance for blood testing.

摘要

背景

血液供者中严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）RNA 血症的频率尚不确定。因此，需要在商业上可用的聚合酶链反应（PCR）系统上对用于检测血液中 SARS-CoV-2 RNA 的 SARS-CoV-2 RNA 检测分析进行验证，以确保数据具有良好的可比性。

研究设计和方法

经许可用于呼吸道拭子 SARS-CoV-2 RNA 检测的 cobas SARS-CoV-2 双靶标逆转录酶 PCR（RT-PCR）分析对用于检测血液中的病毒 RNA 进行了验证。对于验证面板，通过将 SARS-CoV-2 阳性呼吸道标本掺入阴性人血浆中制备 SARS-CoV-2 阳性血浆样本。通过概率分析确定 95%检测限（LOD95）。在临床验证中，纳入了 2019 年冠状病毒病（COVID-19）恢复期血浆（CCP）供者和在重症监护病房（ICU）中治疗的 COVID-19 严重疾病患者。

结果

该 SARS-CoV-2 RT-PCR 分析在血液中的验证表明，其具有高灵敏度、特异性、内和间分析精密度和效率。SARS-CoV-2 RNA 的 LOD95 为靶标 1 的 5.0 基因组拷贝/mL（95%置信区间 [CI]，3.3-12 拷贝/mL）和靶标 2 的 4.3 基因组拷贝/mL（95% CI，2.9-10 拷贝/mL）。在 39 名 CCP 供者的 66 份 CCP 捐赠中，血浆中均未检测到 SARS-CoV-2 RNA。对 19 名 COVID-19 ICU 患者的 25 份血液样本进行筛查，该分析在至少一个靶标中检测到 6 份 SARS-CoV-2 RNA 阳性结果。