Saline collection media and an extraction free workow enables massively scalable, highly sensitive, and cost-effective SARS-CoV-2 testing

The challenges in scaling up SARS-CoV-2 testing capacity include shortages in the supply chain for consumables and reagents. Improvements in consumption patterns can be obtained through removal of key processing steps, including RNA extraction. Here, we present a scalable and validated extraction-free method for the detection of SARS-CoV-2 from swab specimens in saline, with a limit of detection at 1,000 GCE/mL and a sensitivity and specicity of 100%.

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 gammairradiated 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 nal LoD was con rmed 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 con rmation. The extraction-free LoD was con rmed 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 work ow as well as the extraction-free work ow. Cycle quanti cation (Cq) was used to measure viral load and guide the qualitative interpretation of samples. The extraction-free work ow achieved 100% concordance with clinical samples tested with extraction work ow. However, the median Cq of the extraction-free work ow was ~3-4 Cq higher compared to the extraction work ow (Figure 2a). There is a linear correlation of the Cq between the extraction and EF work ows with the exception of two low positive samples on N gene ampli cation ( 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 work ow. 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 work ow 3,6 . In contrast, the method presented here utilizes TBE dilution, heat treatment, and large sample input volumes to achieve 100% sensitivity and speci city 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 extractionbased work ows. Even though the Cqs from the extraction-free work ow are higher than using extracted RNA, the LoD of the extraction-free work ow 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 work ow can achieve the level of accuracy and sensitivity needed for reliable detection of SARS-CoV-2 in clinical samples. The extraction-free work ow using saline transport media removes supply chain constraints, has high accuracy and sensitivity, and it is simple, cost effective and massively scalable. Oropharyngeal Flocked Swab (Affordable IHI) was used in this study. 0.85%-0.90% saline was used as transport media.

Sample Preparation for Limit of Detection and Con rmation Studies
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. Gamma-irradiated 2019 SARS-CoV-2 virus (BEI Resources) was then spiked into the negative matrix at an appropriate concentration and treated at 65°C for 10 minutes. After the deactivation step, 50 µl of the contrived sample was added to 2X volume (100 µl) of 1X TBE buffer (method adopted from Ranoa et. al. 14 ) with 1:200 diluted MS2 internal control (Thermo Fisher, PN A47814), corresponding to a nal MS2 dilution of 1:300 from original stock. Diluted samples were treated at 95°C for 15 minutes prior to RT-qPCR.

Sample preparation for clinical samples
A total of 30 positive for SARS-CoV-2 and 30 negative clinical remnant samples derived from anterior nares swabs collected in normal saline were provided by an independent clinical lab (Laboratory Corporation of America). The samples were stored at -80°C upon initial processing, and were shipped in dry ice to the Helix laboratory. Samples were heat-inactivated at 65°C for 10 min upon receiving. The samples were processed through the Helix COVID-19 Test (EUA2016360), as well as the extraction-free (EF) work ow. A total of 50 µl of each sample was added to 100 µl of 1X TBE with MS2 internal control (1:300 MS2 nal dilution of original stock). For each sample, 14.25 µl was transferred to a new Hard-Shell 384-well PCR plate for lysis at 95°C for 15 minutes. Per sample, 5 µl of One-step RT-PCR mastermix MDX016 (Meridian BioScience) and 0.75 µl of primer/probe mix from TaqPath™ COVID-19 Combo Kit (Thermo Fisher) was added directly to the lysed samples, followed by RT-qPCR at a 20 µl total reaction volume.

RT-PCR setup for extraction-free work ow
The RT-qPCR assay was set up using the TaqPath™ COVID-19 Combo Kit (Thermo Fisher) from Thermo Fisher (EUA200010) and Inhibitor Tolerant 1-Step RT-qPCR Mastermix MDX016 (Meridian BioScience). The cycling conditions are shown in Table 1. The RT-qPCR assay was carried out with the QuantStudio™ 7 Flex Real-Time PCR System. The targeted gene is considered positive if the ampli cation curve crosses the threshold line within 39 quanti cation cycles (Cq < 39) and has a Cq con dence score > 0.8. The nal outcome of the individual sample is based on the number of targets detected as shown in Table 2. Cycle quanti cation (Cq) was used for qualitative interpretation of samples. True positives (TP) were de ned by test results generated using the Helix COVID-19 Test. Sensitivity was de ned as (TP)/(TP+FN) and speci city was de ned as (TN)/(TN+FP). Two or more SARS-CoV-2 targets = POS POS or NEG Valid Positive for SARS-CoV-2 * Criteria to exclude artifacts introduced by low level S gene ampli cation in negative samples, POS with Cq >37 and Cq con dence score >0.8.

Statistical analysis
Differences between extraction and extraction-free Cqs of the clinical positive samples were analyzed using a paired t-test. Correlations between the clinical positive samples extraction and extraction-free Cqs per gene target were analyzed using Spearman's rank test. Analyses were performed with GraphPad