Preanalytical Issues and Cycle Threshold Values in SARS-CoV-2 Real-Time RT-PCR Testing: Should Test Results Include These?
Preanalytical Issues and Cycle Threshold Values in SARS-CoV-2 Real-Time RT-PCR Testing: Should Test Results Include These?
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DOI：
10.1021/acsomega.1c00166
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发表时间：
2021-03-16
期刊：
影响因子：
4.1
通讯作者：
Szunerits S
中科院分区：
文献类型：
--
作者：
Engelmann I;Alidjinou EK;Ogiez J;Pagneux Q;Miloudi S;Benhalima I;Ouafi M;Sane F;Hober D;Roussel A;Cambillau C;Devos D;Boukherroub R;Szunerits S
自从SARS-CoV-2大流行出现以来，全世界的临床实验室都被SARS-CoV-2检测所淹没，该检测使用当前的金标准：实时逆转录聚合酶链反应（RT-PCR）检测。大量疑似病例导致标本运输和RNA提取缓冲液等多种试剂短缺。我们试图提供一些答案，如何强烈的预分析问题影响RT-PCR结果，通过审查不同的运输缓冲液介质和病毒灭活程序的效用，并比较文献数据与我们自己最近的研究结果。我们表明，各种病毒灭活程序和运输缓冲液是可用的，是基于PCR的方法的瓶颈。然而，有效的替代裂解缓冲液仍然更难找到，并且几种快速RT-PCR测定与含胍培养基不相容，使得这方面在当前危机中更具挑战性。此外，具有不同灵敏度的不同SARS-CoV-2特异性RT-PCR试剂盒的可用性使得循环阈值（Ct）值的一般截止水平的定义具有挑战性。只有少数研究考虑了Ct值与病毒感染性的关系以及分析前问题如何影响病毒感染性和RNA检测。我们回顾了目前的数据Ct值和病毒感染性之间的相关性。SARS-CoV-2病毒基因组本身的存在并不足以证明其感染性，在评估样本的感染性时需要谨慎。Ct值和病毒感染性之间的相关性显示，使用实验室开发的靶向RdRp基因的RT-PCR检测，SARS-CoV-2感染性的RT-PCR截止值为34个循环。虽然理想情况下，每个临床实验室都应该进行自己的相关性，但我们相信这篇观点文章可以为其他人提供参考，特别是对COVID-19诊断感兴趣的医生和研究人员，并为协调迈出第一步。
Since the emergence of SARS-CoV-2 pandemic, clinical laboratories worldwide are overwhelmed with SARS-CoV-2 testing using the current gold standard: real-time reverse-transcription polymerase chain reaction (RT-PCR) assays. The large numbers of suspected cases led to shortages in numerous reagents such as specimen transport and RNA extraction buffers. We try to provide some answers on how strongly preanalytical issues affect RT-PCR results by reviewing the utility of different transport buffer media and virus inactivation procedures and comparing the literature data with our own recent findings. We show that various viral inactivation procedures and transport buffers are available and are less of a bottleneck for PCR-based methods. However, efficient alternative lysis buffers remain more difficult to find, and several fast RT-PCR assays are not compatible with guanidine-containing media, making this aspect more of a challenge in the current crisis. Furthermore, the availability of different SARS-CoV-2-specific RT-PCR kits with different sensitivities makes the definition of a general cutoff level for the cycle threshold (Ct) value challenging. Only a few studies have considered how Ct values relate to viral infectivity and how preanalytical issues might affect viral infectivity and RNA detection. We review the current data on the correlation between Ct values and viral infectivity. The presence of the SARS-CoV-2 viral genome in its own is not sufficient proof of infectivity and caution is needed in evaluation of the infectivity of samples. The correlation between Ct values and viral infectivity revealed an RT-PCR cutoff value of 34 cycles for SARS-CoV-2 infectivity using a laboratory-developed RT-PCR assay targeting the RdRp gene. While ideally each clinical laboratory should perform its own correlation, we believe this perspective article could be a reference point for others, in particular medical doctors and researchers interested in COVID-19 diagnostics, and a first step toward harmonization.
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