Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.
Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.
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用于SARS-COV-2检测的开源RNA提取和RT-QPCR方法。
DOI：
10.1371/journal.pone.0246647
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Tjian R
中科院分区：
文献类型：
--
作者：
Graham TGW;Dugast-Darzacq C;Dailey GM;Nguyenla XH;Van Dis E;Esbin MN;Abidi A;Stanley SA;Darzacq X;Tjian R
在COVID-19疫情持续期间，社区重新开放，在全球多个地方引发新一波感染。减轻重新开放的风险将需要广泛的SARS-CoV-2测试，这将大大有助于简单，快速和廉价的测试方法。这项研究评估了几种RNA提取和RT-qPCR的方案，这些方案比流行的方法更简单，更便宜。首先，显示异丙醇沉淀提供了从鼻咽（NP）拭子样品中提取RNA的有效方法。其次，在不进行RNA提取步骤的情况下，评价了NP拭子样本直接添加至RT-qPCR。使用人工拭子样本验证适合直接添加的简单、廉价拭子采集溶液。第三，描述了用于RT-qPCR的开源主混合物，其允许检测NP拭子样本中的病毒RNA，每个反应的检测限约为50个RNA拷贝。来自30个已知阳性临床样品的纯化RNA的定量循环（Cq）值显示该自制主混合物和商业TaqPath主混合物之间的强相关性（r2 = 0.98）。最后，发现终点荧光成像提供准确的诊断读数，而不需要qPCR热循环仪。采用这些简单的开源方法有可能减少COVID-19检测的时间和费用。
Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. This study evaluates several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, isopropanol precipitation is shown to provide an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, direct addition of NP swab samples to RT-qPCRs is evaluated without an RNA extraction step. A simple, inexpensive swab collection solution suitable for direct addition is validated using contrived swab samples. Third, an open-source master mix for RT-qPCR is described that permits detection of viral RNA in NP swab samples with a limit of detection of approximately 50 RNA copies per reaction. Quantification cycle (Cq) values for purified RNA from 30 known positive clinical samples showed a strong correlation (r2 = 0.98) between this homemade master mix and commercial TaqPath master mix. Lastly, end-point fluorescence imaging is found to provide an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, open-source methods has the potential to reduce the time and expense of COVID-19 testing.
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影响因子：
3.7
作者：
Gonzalez-Gonzalez, Everardo;Trujillo-de Santiago, Grissel;Moises Alvarez, Mario
通讯作者：
Moises Alvarez, Mario
影响因子：
5.6
作者：
Dang, C;Jayasena, SD
通讯作者：
Jayasena, SD
影响因子：
19
作者：
Fomsgaard, Anna S.;Rosenstierne, Maiken Worsoe
通讯作者：
Rosenstierne, Maiken Worsoe
DOI：
10.1074/jbc.ra120.015434
发表时间：
2020-11-13
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Mascuch SJ;Fakhretaha-Aval S;Bowman JC;Ma MTH;Thomas G;Bommarius B;Ito C;Zhao L;Newnam GP;Matange KR;Thapa HR;Barlow B;Donegan RK;Nguyen NA;Saccuzzo EG;Obianyor CT;Karunakaran SC;Pollet P;Rothschild-Mancinelli B;Mestre-Fos S;Guth-Metzler R;Bryksin AV;Petrov AS;Hazell M;Ibberson CB;Penev PI;Mannino RG;Lam WA;Garcia AJ;Kubanek J;Agarwal V;Hud NV;Glass JB;Williams LD;Lieberman RL
通讯作者：
Lieberman RL
影响因子：
3.7
作者：
Hasan, Mohammad Rubayet;Mirza, Faheem;Tang, Patrick
通讯作者：
Tang, Patrick