Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection. 2021

Thomas G W Graham, and Claire Dugast-Darzacq, and Gina M Dailey, and Xammy H Nguyenla, and Erik Van Dis, and Meagan N Esbin, and Abrar Abidi, and Sarah A Stanley, and Xavier Darzacq, and Robert Tjian
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California, United States of America.

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.

UI MeSH Term Description Entries
D009305 Nasopharynx The top portion of the pharynx situated posterior to the nose and superior to the SOFT PALATE. The nasopharynx is the posterior extension of the nasal cavities and has a respiratory function. Rhinopharynx,Choanae,Nasopharynges,Nasopharynxes,Rhinopharynges,Rhinopharynxes
D010750 Phosphoproteins Phosphoprotein
D011232 Chemical Precipitation The formation of a solid in a solution as a result of a chemical reaction or the aggregation of soluble substances into complexes large enough to fall out of solution. Precipitation, Chemical
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000086382 COVID-19 A viral disorder generally characterized by high FEVER; COUGH; DYSPNEA; CHILLS; PERSISTENT TREMOR; MUSCLE PAIN; HEADACHE; SORE THROAT; a new loss of taste and/or smell (see AGEUSIA and ANOSMIA) and other symptoms of a VIRAL PNEUMONIA. In severe cases, a myriad of coagulopathy associated symptoms often correlating with COVID-19 severity is seen (e.g., BLOOD COAGULATION; THROMBOSIS; ACUTE RESPIRATORY DISTRESS SYNDROME; SEIZURES; HEART ATTACK; STROKE; multiple CEREBRAL INFARCTIONS; KIDNEY FAILURE; catastrophic ANTIPHOSPHOLIPID ANTIBODY SYNDROME and/or DISSEMINATED INTRAVASCULAR COAGULATION). In younger patients, rare inflammatory syndromes are sometimes associated with COVID-19 (e.g., atypical KAWASAKI SYNDROME; TOXIC SHOCK SYNDROME; pediatric multisystem inflammatory disease; and CYTOKINE STORM SYNDROME). A coronavirus, SARS-CoV-2, in the genus BETACORONAVIRUS is the causative agent. 2019 Novel Coronavirus Disease,2019 Novel Coronavirus Infection,2019-nCoV Disease,2019-nCoV Infection,COVID-19 Pandemic,COVID-19 Pandemics,COVID-19 Virus Disease,COVID-19 Virus Infection,Coronavirus Disease 2019,Coronavirus Disease-19,SARS Coronavirus 2 Infection,SARS-CoV-2 Infection,Severe Acute Respiratory Syndrome Coronavirus 2 Infection,COVID19,2019 nCoV Disease,2019 nCoV Infection,2019-nCoV Diseases,2019-nCoV Infections,COVID 19,COVID 19 Pandemic,COVID 19 Virus Disease,COVID 19 Virus Infection,COVID-19 Virus Diseases,COVID-19 Virus Infections,Coronavirus Disease 19,Disease 2019, Coronavirus,Disease, 2019-nCoV,Disease, COVID-19 Virus,Infection, 2019-nCoV,Infection, COVID-19 Virus,Infection, SARS-CoV-2,Pandemic, COVID-19,SARS CoV 2 Infection,SARS-CoV-2 Infections,Virus Disease, COVID-19,Virus Infection, COVID-19
D000086402 SARS-CoV-2 A species of BETACORONAVIRUS causing atypical respiratory disease (COVID-19) in humans. The organism was first identified in 2019 in Wuhan, China. The natural host is the Chinese intermediate horseshoe bat, RHINOLOPHUS affinis. 2019 Novel Coronavirus,COVID-19 Virus,COVID19 Virus,Coronavirus Disease 2019 Virus,SARS Coronavirus 2,SARS-CoV-2 Virus,Severe Acute Respiratory Syndrome Coronavirus 2,Wuhan Coronavirus,Wuhan Seafood Market Pneumonia Virus,2019-nCoV,2019 Novel Coronaviruses,COVID 19 Virus,COVID-19 Viruses,COVID19 Viruses,Coronavirus 2, SARS,Coronavirus, 2019 Novel,Coronavirus, Wuhan,Novel Coronavirus, 2019,SARS CoV 2 Virus,SARS-CoV-2 Viruses,Virus, COVID-19,Virus, COVID19,Virus, SARS-CoV-2,Viruses, COVID19
D000086462 Coronavirus Nucleocapsid Proteins Nucleocapsid proteins from species of the CORONAVIRIDAE virus family. N Protein, Coronavirus,Nucleocapsid Protein, Coronavirus,Coronavirus N Protein,Coronavirus Nucleocapsid Protein,Nucleocapsid Proteins, Coronavirus
D000087123 COVID-19 Nucleic Acid Testing Diagnosis of COVID-19 by assaying bodily fluids or tissues for the presence of the VIRAL RNA of SARS-COV-2. COVID-19 RT-PCR Testing,COVID19 Nucleic Acid Testing,SARS Coronavirus 2 Nucleic Acid Testing,SARS Coronavirus 2 RT-PCR Testing,SARS-CoV-2 Infection Nucleic Acid Testing,SARS-CoV2 RT-PCR Testing,COVID 19 Nucleic Acid Testing,COVID 19 RT PCR Testing,COVID-19 RT-PCR Testings,RT-PCR Testing, COVID-19,RT-PCR Testing, SARS-CoV2,SARS CoV 2 Infection Nucleic Acid Testing,SARS CoV2 RT PCR Testing,SARS Coronavirus 2 RT PCR Testing,SARS-CoV2 RT-PCR Testings,Testing, COVID-19 RT-PCR,Testing, SARS-CoV2 RT-PCR
D012367 RNA, Viral Ribonucleic acid that makes up the genetic material of viruses. Viral RNA
D057230 Limit of Detection Concentration or quantity that is derived from the smallest measure that can be detected with reasonable certainty for a given analytical procedure. Limits of Detection,Detection Limit,Detection Limits

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