Multiplexed and Extraction-Free Amplification for Simplified SARS-CoV-2 RT-PCR Tests

Abstract

The rapid onset of the global COVID-19 pandemic has led to challenges for accurately diagnosing the disease, including supply shortages for sample collection, preservation, and purification. Currently, most diagnostic tests require RNA extraction and detection by RT-PCR; however, extraction is expensive and time-consuming and requires technical expertise. With these challenges in mind, we report extraction-free, multiplexed amplification of SARS-CoV-2 RNA from 246 clinical samples, resulting in 86% sensitivity and 100% specificity. The multiplex RT-PCR uses the CDC singleplex targets and has an LoD of 2 c/μL. We also report on amplification using a range of master mixes in different transport media. This work can help guide which combinations of reagents will enable accurate results when availability of supplies changes throughout the pandemic. Implementing these methods can reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.

Evaluation of extraction-free RT-qPCR methods for SARS-CoV-2 diagnostics

“…For instance, Burton et al [ 22 ] did not register any significant differences on implementing heating regimens of 56/60°C. In sum, in line with the available evidence [ 9 , 15 , 19 – 24 ], our results support the use of EF techniques in routine practice. The heat treatment procedure introduced in order to lyse epithelial cells may, however, reduce sensitivity.…”

“…Several EF protocols have recently been described and validated [ 9 , 15 , 19 – 24 ]. Across these studies, the relative sensitivity of EF techniques has been seen to vary from 55% to >95%.…”

“…All these samples were deemed positive according to the traditional extraction-based (EB) RT-qPCR technique. During an internal discussion, it was decided that a sample size of 100 positive samples would be sufficiently powered to determine a sensitivity of 90% [ 15 , 16 ] provided by the RNA extraction-free (EF) methods. It was assumed that the specificity of the EF method was 100%.…”

Clinical validation of an open-access SARS-COV-2 antigen detection lateral flow assay, compared to commercially available assays

“…RNA is extracted from nasal swab samples using the QIAamp Viral Mini Kit as recommended by the CDC. The extracted RNA is amplified using a multiplexed version of the CDC 2019-nCoV Real-Time RT-qPCR targets (three targets: N1, N2, and RP) with the QuantaBio qScript XLT 1-step mix on a BioRad CFX96 Real-Time PCR Detection System [ 9 ]. The N1 gene is used to quantify SARS-CoV-2 viral load and the RP gene is used to quantify human RNA load.…”

Rapid and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus 2 in Label-Free Manner Using Micromechanical Sensors

“…Therefore, methodological simplification is crucial to control infection by increasing availability and efficiency. Several efforts have been made to circumvent RNA extraction in COVID-19 RT-PCR testing [ 9 , 10 , 11 , 12 ]. However, the presented methods still require DNA amplification, which is a major bottleneck in this procedure.…”