With sufficient data that suggest SARS-CoV-2 can be spread by pre-symptomatic/asymptomatic carriers1, large-scale and repeated testing for SARS-CoV-2 infection is critical to control the spread of COVID-192. The standard workflow often includes total RNA extraction followed by real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). One of the challenges in scaling up SARS-CoV-2 testing capacity includes shortages in the supply chain for consumables and reagents. Therefore, reduced consumption can be achieved through removal of key processing steps such as RNA extraction.
An extraction-free workflow is less laborious than a RNA extraction workflow, but it is also less robust and prone to failure due to reaction inhibition by specimen components3. Studies suggested that dry swabs 4,5 and swab samples stored in appropriate transport media such as Universal/Viral Transport Media (UTM/VTM) 3,5–10 or water 6 can be tested by RT-qPCR without the need for RNA extraction. The general workflow includes a lysis step, incubating samples between 70°C to 99°C for 5-15 minutes, followed by RT-qPCR setup using a small amount of sample and specific RT-qPCR mastermixes. Only one of these studies8 reported a limit of detection (LoD), which is greater than 6,000 genomic copies equivalent/mL (GCE/mL) and it was high for a PCR-based assay 11. In addition, the increased demand for testing has constrained the supply of UTM/VTM. Alternative transport media and, at the very least, an end-to-end protocol with comparable sensitivity to extraction-based methods is essential to alleviating possible trade-offs between efficiency and sensitivity.
Saline is easily obtainable and most importantly has demonstrated stability and usability in swab-based sampling 12. Saline transport media is a mixture of salt (sodium chloride) and water at a pH of 4.5-7.0, which is similar to the sodium concentration of human bodily fluids. Saline has been shown to perform poorly in an extraction-free workflow when the sample is added directly into the RT-qPCR reaction 3,6. However, the workflow described here was adapted from a saliva-based SARS-CoV-2 molecular test 8 at which the sample is first diluted with 1X TBE ((Tris/Borate/EDTA Buffer) and then treated with heat at 95°C for 15 minutes. The heating step lysed the viral particles, and is also likely to inactivate the inhibitory components in the sample, therefore allowing better sensitivity in detecting SARS-CoV-2 nucleic acid. The method is further optimized to be used for processing saline-based swab specimens.
To establish the limit of detection (LoD) of the assay, contrived samples were generated from upper respiratory specimens negative for SARS-CoV-2 collected via anterior nares swab in 0.85% saline solution. The samples were pooled to obtain a large volume of negative matrix and spiked with gamma-irradiated 2019 SARS-CoV-2 virus (BEI Resources) at an appropriate concentration. Triplicates were screened at each concentration of inactive virus, ranging from 4,000 GCE/mL to 250 GCE/mL (Figure 1). The final LoD was confirmed using 20 replicates. All three replicates were called positive at 500 GCE/mL during LoD screening; however, only 16/20 samples were called positive at 500 GCE/mL during confirmation. The extraction-free LoD was confirmed at 1,000 GCE/mL with 20/20 positive samples.
To evaluate the performance of extraction-free assay on clinical samples, this study used 30 positive and 30 negative remnant clinical swab samples in saline provided by an independent clinical lab. The samples were processed through the Helix standard extraction workflow as well as the extraction-free workflow. Cycle quantification (Cq) was used to measure viral load and guide the qualitative interpretation of samples. The extraction-free workflow achieved 100% concordance with clinical samples tested with extraction workflow. However, the median Cq of the extraction-free workflow was ~3-4 Cq higher compared to the extraction workflow (Figure 2a). There is a linear correlation of the Cq between the extraction and EF workflows with the exception of two low positive samples on N gene amplification (Figure 2b).
Here, we presented a massively scalable, highly sensitive and cost effective method in detecting SARS-CoV-2 for SARS-CoV-2 testing using saline collection media. The method outlined here consists of a minimal number of steps and utilizes a standard qPCR assay downstream, thus allowing direct implementation into the existing workflow. Saline has demonstrated usability in swab-based sampling 12 and is easily obtainable. Saline can be stored at room temperature. Upon sample collection, the sample is stable for up to 54 hours without special storage conditions 12. However, saline-based specimens have been shown to perform poorly in extraction-free workflow 3,6. In contrast, the method presented here utilizes TBE dilution, heat treatment, and large sample input volumes to achieve 100% sensitivity and specificity in 60 clinical samples, and a LoD at 1,000 GCE/mL, which is comparable to assays using extraction-based methods 11,13. The limitations of this study include the relatively small number of available clinical samples that preclude a more thorough analysis of sensitivity compared to extraction-based workflows. Even though the Cqs from the extraction-free workflow are higher than using extracted RNA, the LoD of the extraction-free workflow is equivalent, and detection of SARS-CoV-2 in clinical samples is highly correlated between the two methods. Our results suggest that a properly validated extraction-free RT-qPCR workflow can achieve the level of accuracy and sensitivity needed for reliable detection of SARS-CoV-2 in clinical samples. The extraction-free workflow using saline transport media removes supply chain constraints, has high accuracy and sensitivity, and it is simple, cost effective and massively scalable.