Simple, Inexpensive RNA Isolation and One‐Step RT‐qPCR Methods for SARS‐CoV‐2 Detection and General Use

Abstract

The most common method for RNA detection involves reverse transcription followed by quantitative polymerase chain reaction (RT‐qPCR) analysis. Commercial one‐step master mixes—which include both a reverse transcriptase and a thermostable polymerase and thus allow performing both the RT and qPCR steps consecutively in a sealed well—are key reagents for SARS‐CoV‐2 diagnostic testing; yet, these are typically expensive and have been affected by supply shortages in periods of high demand. As an alternative, we describe here how to express and purify Taq polymerase and M‐MLV reverse transcriptase and assemble a homemade one‐step RT‐qPCR master mix. This mix can be easily assembled from scratch in any laboratory equipped for protein purification. We also describe two simple alternative methods to prepare clinical swab samples for SARS‐CoV‐2 RNA detection by RT‐qPCR: heat‐inactivation for direct addition, and concentration of RNA by isopropanol precipitation. Finally, we describe how to perform RT‐qPCR using the homemade master mix, how to prepare in vitro−transcribed RNA standards, and how to use a fluorescence imager for endpoint detection of RT‐PCR amplification in the absence of a qPCR machine In addition to being useful for diagnostics, these versatile protocols may be adapted for nucleic acid quantification in basic research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of a one‐step RT‐qPCR master mix using homemade enzymes Basic Protocol 2: Preparation of swab samples for direct RT‐PCR Alternate Protocol 1: Concentration of RNA from swab samples by isopropanol precipitation Basic Protocol 3: One‐step RT‐qPCR of RNA samples using a real‐time thermocycler Support Protocol: Preparation of RNA concentration standards by in vitro transcription Alternate Protocol 2: One‐step RT‐PCR using endpoint fluorescence detection

A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR

“…There are a large number of commercially available kits for cDNA synthesis which are very expensive. Here, we have used a protocol for synthesis and purification of Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Graham et al 2021 ) which was used for the synthesis of cDNA. Briefly, expression plasmid pET-28a_6H-MMLV_RT_D524N-6H (Addgene plasmid # 166945) was transformed into BL21 competent cells.…”

“…Proteins fractions were eluted in storage buffer and stored at −80°C until further use. (For buffer compositions refer materials and methods, and for detailed purification protocol refer (Graham et al 2021 )). cDNA synthesis was carried using 1µg of RNA from mouse ESCs as described in materials and methods.…”

“…Next, cDNA was diluted to 1:10 v/v and used as template for real-time PCR. Hot-start Taq polymerase was purified as per the previously published protocol (Graham et al 2021 ). Briefly, expression plasmid pET-28a_6H-TAQ_E602D (Addgene #166944) was transformed into BL21 competent cells, cultures were grown overnight at 37°C, induced with 1 mM IPTG, pelleted down and flash-frozen in liquid nitrogen and stored at −80°C until further use.…”

“…Pellets were resuspended in lysis buffer and subjected to Ni-NTA based purification followed by HiTrap heparin purification. Proteins fractions were eluted in storage buffer and stored at −80°C until further use (for buffer composition refer materials and methods and for detailed purification protocol refer (Graham et al 2021 )). This hot-start Taq polymerase was used in the preparation of the in-house PCR mastermix (figure 1 D and E ).…”

“…Hence, no changes are required in the optical settings of the instrument while using EvaGreen dye. Here, we demonstrate a protocol to purify in-house Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Graham et al 2021 ) for cDNA synthesis and designed an in-house real-time PCR mastermix using EvaGreen or SYBR Green I dye and in-house Hot start Taq DNA polymerase. The Ct values, amplification plots, dissociation curves were comparable between commercially available SYBR Green I PCR mastermix, and our in-house real-time PCR mastermix with either SYBR Green I or Evagreen dye.…”