SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP

Abstract

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

Considerations in Real-time Reverse Transcription Polymerase Chain Reaction (rRT-PCR) for the Detection of SARS-CoV-2 from Nasopharyngeal Swabs

“…Before taking the extraction plate into the hood, mark the plate number on the side of the plate, so that the number is visible. The sealing foil/wrap of the extraction plate should be removed inside the cabinet [10].…”

“…These items should be arranged in the same order in a unidirectional manner to avoid cross-contamination. Each item that is taken inside the cabinet should be thoroughly cleaned with 10% sodium hypochlorite, distilled water, and 70% ethanol if it is required [10]. Before beginning to add the samples to the wells in the extraction, ensure that the work materials are arranged in a unidirectional flow either from left to right or from right to left depending on the convenience of the personnel.…”

“…Discard the tissue in the discard container. If during transfer of sample, a spillage occurs on the work area then immediately follow the same cleaning protocol -10% sodium hypochlorite, followed by distilled water, then 70% alcohol [10].…”

“…The extraction plates should be carefully taken out and placed in a biosafety cabinet for the transfer of RNA. RNA can be transferred into PCR strip tubes and placed at 40°C (for 1 hour) until used for setting up PCR, or it can be directly transferred into the PCR tubes (after addition of master mix) and the rRT-PCR can be set up immediately [10][11][12].…”

Special Issue “Emerging Viruses 2020: Surveillance, Prevention, Evolution and Control”

“…In the face of the current pandemic, many published manuscripts were related to SARS-CoV-2. Klein et al [2] presented a magnetic bead-based protocol for SARS-CoV-2 RNA extraction that used less expensive devices but yielded RNA extracts comparable to the commercially available QIAcube viral RNA extraction kit, providing similar sensitivity and specificity by the commonly applied detection methods RT-qPCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP), using E and N targets, respectively. Stoddard et al [3] described an ensemble molecular docking approach and proposed a designed guideline to virtually trial potential drug candidates to the SARS-CoV-2 main protease.…”