Inexpensive, versatile and open-source methods for SARS-CoV-2 detection

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited a second wave 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. To this end, we evaluated several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, we show that isopropanol precipitation provides an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, we evaluate direct addition of NP swab samples to RT-qPCR reactions without an RNA extraction step. We describe a simple, inexpensive swab collection solution suitable for direct addition, which we validate using contrived swab samples. Third, we describe an open-source master mix for RT-qPCR and show that it permits detection of viral RNA in NP swab samples. Lastly, we show that an end-point fluorescence measurement provides an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, inexpensive methods has the potential to significantly reduce the time and expense of COVID-19 testing.

[1]  R. Tjian,et al.  Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection , 2020, RNA.

[2]  Milind Tambe,et al.  Test sensitivity is secondary to frequency and turnaround time for COVID-19 surveillance , 2020, medRxiv : the preprint server for health sciences.

[3]  Kenneth S. Kosik,et al.  A Fast and Accessible Method for the Isolation of RNA, DNA, and Protein To Facilitate the Detection of SARS-CoV-2 , 2020, Journal of Clinical Microbiology.

[4]  Paras Jain,et al.  Multiplexed and Extraction-Free Amplification for Simplified SARS-CoV-2 RT-PCR Tests , 2020, Analytical chemistry.

[5]  Z. Zhao,et al.  A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of SARS-CoV-2 , 2020, bioRxiv.

[6]  S. Jayasena,et al.  Oligonucleotide inhibitors of Taq DNA polymerase facilitate detection of low copy number targets by PCR. , 1996, Journal of molecular biology.

[7]  R. González-Montelongo,et al.  Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples , 2020, International Journal of Infectious Diseases.

[8]  K. Schallenberger,et al.  Pre-treatment of the clinical sample with Proteinase K allows detection of SARS-CoV-2 in the absence of RNA extraction , 2020, bioRxiv.

[9]  A. Ellington,et al.  A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production , 2020, bioRxiv.

[10]  Comparison of 12 molecular detection assays for SARS-CoV-2 , 2020 .

[11]  D. Marino,et al.  Fast SARS-CoV-2 detection protocol based on RNA precipitation and RT-qPCR in nasopharyngeal swab samples , 2020, medRxiv.

[12]  A. Fomsgaard,et al.  An alternative workflow for molecular detection of SARS-CoV-2 – escape from the NA extraction kit-shortage, Copenhagen, Denmark, March 2020 , 2020, medRxiv.

[13]  P. Grant,et al.  Extraction-free COVID-19 (SARS-CoV-2) diagnosis by RT-PCR to increase capacity for national testing programmes during a pandemic , 2020, bioRxiv.

[14]  X. Cui,et al.  One-step RNA extraction for RT-qPCR detection of 2019-nCoV , 2020, bioRxiv.

[15]  Devon Chandler-Brown,et al.  A Highly Scalable and Rapidly Deployable RNA Extraction-Free COVID-19 Assay by Quantitative Sanger Sequencing , 2020, bioRxiv.

[16]  B. Reinius,et al.  Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR , 2020, Nature communications.

[17]  P. Tang,et al.  High-throughput extraction of SARS-CoV-2 RNA from nasopharyngeal swabs using solid-phase reverse immobilization beads , 2020, medRxiv.

[18]  A. Ellington,et al.  High-Surety Isothermal Amplification and Detection of SARS-CoV-2 , 2020, bioRxiv.

[19]  Megan L Hochstrasser,et al.  Blueprint for a pop-up SARS-CoV-2 testing lab , 2020, Nature Biotechnology.

[20]  Itzel Montserrat Lara-Mayorga,et al.  Portable and accurate diagnostics for COVID-19: Combined use of the miniPCR thermocycler and a well-plate reader for SARS-CoV-2 virus detection , 2020, medRxiv.

[21]  H. Yassine,et al.  Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA , 2020, medRxiv.

[22]  Scott Tighe,et al.  Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step , 2020, PLoS biology.

[23]  C. Liu,et al.  SalivaDirect: Simple and sensitive molecular diagnostic test for SARS-CoV-2 surveillance , 2020, medRxiv.

[24]  S. Goff,et al.  RNase H domain mutations affect the interaction between Moloney murine leukemia virus reverse transcriptase and its primer-template. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Geneviève Pépin,et al.  SARS-CoV-2 detection by direct rRT-PCR without RNA extraction , 2020, Journal of Clinical Virology.

[26]  E. Yap,et al.  Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler , 2020, bioRxiv.

[27]  Vinayak Agarwal,et al.  A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits , 2020, The Journal of Biological Chemistry.

[28]  R. Rasmussen.  Quantification on the LightCycler , 2001 .

[29]  R. Soto-Rifo,et al.  SARS-CoV-2 detection from nasopharyngeal swab samples without RNA extraction , 2020, bioRxiv.

[30]  Rodrigo A. Gutiérrez,et al.  A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR , 2020, Scientific Reports.

[31]  C. Schneider,et al.  A streamlined approach to rapidly detect SARS-CoV-2 infection, avoiding RNA extraction , 2020, medRxiv.

[32]  A. Chenchik,et al.  TaqStart Antibody: "hot start" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. , 1994, BioTechniques.

[33]  Hyeshik Chang,et al.  Development of a Laboratory-safe and Low-cost Detection Protocol for SARS-CoV-2 of the Coronavirus Disease 2019 (COVID-19) , 2020, Experimental neurobiology.

[34]  N. Xia,et al.  Room-temperature-storable PCR mixes for SARS-CoV-2 detection , 2020, bioRxiv.

[35]  M. Rieder,et al.  Preliminary support for a "dry swab, extraction free" protocol for SARS-CoV-2 testing via RT-qPCR. , 2020, bioRxiv : the preprint server for biology.

[36]  Season Wong,et al.  The Potential Use of Unprocessed Sample for RT-qPCR Detection of COVID-19 without an RNA Extraction Step , 2020, bioRxiv.

[37]  D. Birch,et al.  Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. , 1992, Nucleic acids research.