COVID-19 diagnostics for resource-limited settings: Evaluation of "unextracted" qRT-PCR
COVID-19 diagnostics for resource-limited settings: Evaluation of "unextracted" qRT-PCR
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DOI：
10.1002/jmv.26328
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发表时间：
2020-07-28
影响因子：
12.7
通讯作者：
Schmitz, Jonathan E.
中科院分区：
文献类型：
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作者：
Adams, Nicholas M.;Leelawong, Mindy;Schmitz, Jonathan E.
2019年冠状病毒病（COVID-19）大流行导致对分子诊断的需求急剧增加。不幸的是，获得RNA提取试剂可能是基于定量实时逆转录酶-聚合酶链反应（qRT-PCR）的方法的瓶颈，这是由于非凡的供应链压力和病毒在全球范围内进入资源有限的环境。为了为此类环境提供灵活的诊断选择，我们在此报告了使用疾病控制中心（CDC）广泛使用的严重急性呼吸综合征冠状病毒2型（N1/N2/N3靶向病毒核衣壳和RP对照靶向人RNase P）的引物/探针组对qRT-PCR进行的“未提取修饰”。该方法用病毒转运培养基（VTM）的热灭活步骤代替RNA提取/纯化，然后直接接种（有或没有VTM离心浓缩）到PCR主混合物中。使用我们临床工作流程中的护理衍生物，我们比较了传统和未提取的CDC方法。尽管分析灵敏度明显下降，（通过较高的C（t）值），特别是对于N2引物/探针组，我们观察到N1的提取和未提取结果之间的分类阳性一致性较高（未浓缩VTM-38/40;浓缩VTM-39/41），N3（未浓缩的VTM-38/40;浓缩的VTM-41/41）和RP（未浓缩的和浓缩的VTM-81/81）。N1/N2/N3的阴性分类一致性也很高。总体而言，这些结果表明，实验室可以调整和验证未提取的qRT-PCR方案，作为克服供应限制的应急措施，对分类结果的影响最小。
The coronavirus disease 2019 (COVID-19) pandemic has created a precipitous increase in the need for molecular diagnostics. Unfortunately, access to RNA extraction reagents can represent a bottleneck for quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR)-based methodologies, stemming from both extraordinary supply-chain stresses and the global reach of the virus into resource-limited settings. To provide flexible diagnostic options for such environments, we report here an "unextracted modification" for qRT-PCR using the Centers for Disease Control's (CDC's) widely utilized primers/probe sets for severe acute respiratory syndrome coronavirus 2 (N1/N2/N3 targeting viral nucleocapsid and RP-control targeting human RNase P). This approach replaces RNA extraction/purification with a heat-inactivation step of viral transport media (VTM), followed by direct inoculation-with or without VTM spin concentration-into PCR master mixes. Using derivatives of care from our clinical workflow, we compared traditional and unextracted CDC methodologies. Although some decrease in analytic sensitivity was evident (by higherC(t)values) without extraction, in particular for the N2 primer/probe-set, we observed high categorical positive agreement between extracted and unextracted results for N1 (unconcentrated VTM-38/40; concentrated VTM-39/41), N3 (unconcentrated VTM-38/40; concentrated VTM-41/41), and RP (unconcentrated and concentrated VTM-81/81). The negative categorical agreement for N1/N2/N3 was likewise high. Overall, these results suggest that laboratories could adapt and validate unextracted qRT-PCR protocols as a contingency to overcome supply limitations, with minimal impact on categorical results.