Abstract
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.
Highlights
The current global pandemic of SARS-CoV-2 has infected an estimated 98 million people worldwide and claimed over 2.1 million lives (Worldometer, https://www.worldometers.info/ coronavirus/, accessed 1-22-2021)
The U.S Centers for Disease Control and Prevention “gold standard” test for COVID-19 detects SARS-CoV-2 viral RNA purified from patient nasopharyngeal swabs
The samples were first inactivated in one of two ways: Samples Pos1-Pos2 and Neg1-Neg2 were mixed with 1 volume of 2x DNA/RNA Shield, while the remaining samples (Neg3-Neg12 and Pos3-Pos12) were treated with 0.4 mg/mL proteinase K for 30 min at 37 ̊C, heated at 95 ̊C for 5 min, and incubated at 75 ̊C for 30 min
Summary
The current global pandemic of SARS-CoV-2 has infected an estimated 98 million people worldwide and claimed over 2.1 million lives (Worldometer, https://www.worldometers.info/ coronavirus/, accessed 1-22-2021). The true number of cases is likely to be even higher, and a full understanding of the scope of the pandemic has been hindered by a persistent lack of widespread testing. The U.S Centers for Disease Control and Prevention “gold standard” test for COVID-19 detects SARS-CoV-2 viral RNA purified from patient nasopharyngeal swabs. Researchers and clinicians aiming to implement RT-PCR testing for COVID19 have faced a shortage of the necessary reagents to perform tests in addition to the long. Analysis, decision to publish, or preparation of the manuscript
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