Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR.
Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR.
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DOI：
10.1371/journal.pone.0247792
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Levi V
中科院分区：
文献类型：
--
作者：
Genoud V;Stortz M;Waisman A;Berardino BG;Verneri P;Dansey V;Salvatori M;Remes Lenicov F;Levi V
实时逆转录PCR（RT-qPCR）是检测鼻咽拭子标本中严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）的金标准技术。通过RT-qPCR的分析通常需要先前的提取步骤以获得纯化的病毒RNA。不幸的是，RNA提取在许多国家构成了早期检测的瓶颈，因为它昂贵、耗时并且取决于商业试剂盒的可用性。在这里，我们描述了一种用于通过RT-qPCR从盐水溶液中的鼻咽拭子临床样本中检测SARS-CoV-2的无提取方案。该方法包括用蛋白酶K处理，然后热灭活（PK+HID方法）。我们证明，与热灭活程序相比，PK+HID提高了RT-qPCR的性能。此外，我们表明这种无提取方案可以与多种多重RT-qPCR试剂盒组合。该方法结合针对N和ORF 1ab基因的多重检测试剂盒对106份阳性和106份阴性临床样本的分析显示，灵敏度为0.99，特异性为0.99。总之，PK+HID是用于SARS-CoV-2的无提取RT-qPCR测定的稳健、快速和廉价的方法。阿根廷国家药品、食品和医疗器械管理局最近批准使用这种方法。
Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.
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影响因子：
16.6
作者：
Smyrlaki I;Ekman M;Lentini A;Rufino de Sousa N;Papanicolaou N;Vondracek M;Aarum J;Safari H;Muradrasoli S;Rothfuchs AG;Albert J;Högberg B;Reinius B
通讯作者：
Reinius B
影响因子：
12.7
作者：
Adams, Nicholas M.;Leelawong, Mindy;Schmitz, Jonathan E.
通讯作者：
Schmitz, Jonathan E.
影响因子：
9.4
作者：
ESPY, MJ;PATEL, R;SMITH, TF
通讯作者：
SMITH, TF
DOI：
10.1016/j.jcv.2020.104423
发表时间：
2020-07-01
期刊：
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology
影响因子：
--
作者：
Merindol, Natacha;Pepin, Genevieve;Danylo, Alexis
通讯作者：
Danylo, Alexis
影响因子：
14.9
作者：
Ruijter JM;Ramakers C;Hoogaars WM;Karlen Y;Bakker O;van den Hoff MJ;Moorman AF
通讯作者：
Moorman AF