DNA Cross-Reactivity of the CDC-Specified SARS-CoV-2 Specimen Control Leads to Potential for False Negatives and Underreporting of Viral Infection.
DNA Cross-Reactivity of the CDC-Specified SARS-CoV-2 Specimen Control Leads to Potential for False Negatives and Underreporting of Viral Infection.
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CDC 指定的 SARS-CoV-2 样本对照的 DNA 交叉反应性可能导致假阴性和漏报病毒感染。
DOI：
10.1093/clinchem/hvaa284
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发表时间：
2021
影响因子：
9.3
通讯作者：
Rosebrock,AdamP
中科院分区：
文献类型：
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作者：
Rosebrock,AdamP
CDC 2019-nCoV实时RTPCR诊断试剂盒由3种qPCR检测试剂组成：N1和N2从逆转录病毒RNA中生成SARS-CoV-2特异性扩增子; RP是一种靶向人RPP 30基因内单个外显子的人标本和提取对照。该对照品的失败旨在表明RNA的潜在损失或RNA降解（1）。在实践中，这种设计在排除RNA病毒检测的情况下会产生误导性的对照阳性信号。CDC RP质控品能够检测逆转录的RNA;也可检测人基因组DNA。事实上，CDC使用相同的对照设计用于分别靶向RNA和DNA基因组的病毒呼吸道病原体的RT-qPCR和qPCR面板（与CDC的个人交流）。当存在人DNA时，不需要完整的RNA和逆转录来产生阳性标本和提取对照信号。使用CDC设计，基因组DNA的单位拷贝足以产生强对照信号（数据未显示）。通过用于从临床样本中分离RNA的固相和液-液提取程序共纯化DNA。从COVID-19病例鼻咽拭子中提取的RNA样本的仅qPCR（无RT）再分析在所有检测标本中产生了强对照信号（数据未显示）。更令人担忧的是，DNA交叉反应性导致真阳性患者样本的分析假阴性，其中RNA已被降解（图1）。在分析之前合并多个样品用于增加通量并降低测试成本（2-4）。由DNA反应性对照引起的问题通过合并而被放大：一个含有RNA酶的样品可以使整个合并的病毒RNA呈阴性，而来自单个患者的几个细胞的DNA就足以产生标本
CDC 2019-nCoV Real-Time RTPCR Diagnostic Panel is comprised of 3 qPCR assays: N1 and N2 generate SARS-CoV-2-specific amplicons from reverse transcribed viral RNA; RP is a human specimen and extraction control that targets a single exon within the human RPP30 gene. Failure of this control is intended to indicate potential loss of RNA or RNA degradation (1). In practice, this design generates a misleading control-positive signal in scenarios that preclude RNA virus detection. The CDC RP control is capable of detecting reverse transcribed RNA; it also detects human genomic DNA. Indeed, the CDC uses the same control design for RT-qPCR and qPCR panels targeting viral respiratory pathogens with RNA and DNA genomes, respectively (personal communication with CDC). When human DNA is present, intact RNA and reverse transcription are unnecessary to generate a positive specimen and extraction control signal. Single-digit copies of genomic DNA are sufficient to generate a strong control signal using the CDC design (data not shown). DNA is copurified by solid phase and liquid–liquid extraction procedures used for isolation of RNA from clinical specimens. qPCR-only (no-RT) reanalysis of RNA samples extracted from COVID-19 case nasopharyngeal swabs yielded strong control signals from all specimens tested (data not shown). More worryingly, DNA cross-reactivity leads to analytical false negatives from true-positive patient samples where RNA has been degraded (Fig. 1). Pooling multiple samples prior to analysis is being used to increase throughput and reduce testing cost (2–4). The problems caused by a DNA-reactive control are magnified by pooling: one RNase-containing sample can render an entire pool virus RNA negative, while a few cells’ worth of DNA from a single patient are sufficient to generate a specimen