Direct RT-PCR amplification of SARS-CoV-2 from clinical samples using a concentrated viral lysis-amplification buffer prepared with IGEPAL-630

The pandemic of 2019 caused by the novel coronavirus (SARS-CoV-2) is still rapidly spreading worldwide. Nucleic acid amplification serves as the gold standard method for confirmation of COVID-19 infection. However, challenges faced for diagnostic laboratories from undeveloped countries includes shortage of kits and supplies to purify viral RNA. Therefore, it is urgent to validate alternative nucleic acid isolation methods for SARS-CoV-2. Our results demonstrate that a concentrated viral lysis amplification buffer (vLAB) prepared with the nonionic detergent IGEPAL enables qualitative detection of SARS-CoV-2 by direct Reverse Transcriptase-Polymerase Chain Reaction (dRT-PCR). Furthermore, vLAB was effective in inactivating SARS-CoV-2. Since this method is inexpensive and no RNA purification equipment or additional cDNA synthesis is required, this dRT-PCR with vLAB should be considered as an alternative method for qualitative detection of SARS-CoV-2.


Introduction
Severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) has generated a global pandemic due its rapid spread (~57 million con rmed cases) and fatal progression (~1.4 million deaths) of the coronavirus infection disease (COVID-19) (1). Early diagnosis of COVID-19 is crucial for disease treatment and control (2). Many symptoms of SARS-CoV-2 overlap with other respiratory illnesses, so con rmation of the presence of the virus is necessary for accurate diagnosis (3). Currently, the reverse transcriptase ampli cation (RT-PCR) method is the gold standard for the diagnosis of SARS-CoV-2 in respiratory samples (4). In early 2020, the Centers for Disease Control and Prevention (CDC) in the USA developed an RT-PCR assay for detection of SARS-CoV-2 (5), and a few weeks later the Federal Drug Administration (FDA) authorized this assay for emergency use (5,6). The RT-PCR primers described in the CDC protocol showed high sensitivity (600−1200 viral genome copies/mL) and speci city (7) so are routinely used in many laboratories around the world. This assay requires RNA puri cation and several puri cation kits have been validated. However, the high demand for RNA puri cation kits has resulted in worldwide shortages that have affected several diagnostic laboratories, especially in undeveloped countries. Therefore, it is necessary to validate new RNA isolation methods useful for COVID detection.
Direct PCR (dPCR) is a method of DNA/RNA ampli cation directly from a sample without performing DNA/RNA isolation and puri cation steps. As such, this technique greatly reduces sample processing time (8). Detergents included in dPCR buffers induce cellular lysis with release of nucleic acids, which allows robust ampli cation despite the presence of PCR inhibitors often found in crude samples (9,10).
IGEPAL CA-630 is a nonionic, non-denaturing detergent that has been successfully used for dRT-PCR ampli cation of in uenza virus (10). However, IGEPAL has not been validated for detection of SARS-CoV-2 in clinical samples. Here, we used IGEPAL to prepare a viral lysis-ampli cation buffer (vLAB) and demonstrated that this buffer is suitable for the detection of SARS-CoV-2 by dRT-PCR in clinical samples following the CDC protocol.

Methods
All methods were carried out in accordance with guidelines and regulations in methods section.  We infected monolayers of Vero E6 cells with mNG-SARS-Cov2 in vLAB to evaluate virus inactivation. For these experiments, 10 μL of mNG-SARS-CoV-2 (1x10 7 PFU/mL) were added to 90 μL of vLAB. Some samples were incubated at room temperature (RT) for 10 minutes, and other samples were incubated at RT for 20 minutes. Post-incubation, 10 μL from each sample was added to each well of a Falcon® 12well plate seeded with 5x10 4 cells/well in 2 mL Gibco 1X MEM with 10% fetal bovine serum and gentamycin. Each condition (negative control, positive control, 10 minutes, and 20 minutes) was run in triplicate. Plates were incubated at 37°C with 5% CO 2 for 3 days and then analyzed by uorescent microscopy. To con rm inactivation, from the rst passage plate, 10 μL of supernatant was inoculated in triplicate onto another 12-well plate seeded with 5x10 4 cells/well in 2 mL of 10% 1X MEM. This plate was incubated again at 37°C for 3 days and then analyzed by microscopy as before. Inactivation experiments were conducted in BSL-3 facilities (Dr. Bukreyev's laboratory at UTMB) and the inactivation protocol was approved by the Institutional Biosafety Committee at UTMB. dPCR from pure SARS-Cov-2 RNA and inactivated cells.
For these experiments, we used SARS-Cov-2 RNA (ranging from 0-50000 copies) obtained from WRCEVA or supernatants of infected cells previously inactivated with vLAB as described above. We conducted all RT-PCR ampli cations with the Quantabio RT-qPCR Tough Mix Kit and we used primers and probes for nucleocapsid (N1 and N2) designed by CDC included in the 2019-nCov CDC EUA Kit, 1000 rxn (Integrated DNA Technologies, Coralville, Iowa). After adding template (2 µl) to RT-PCR master mix, reaction (20 µl total volume) was transferred to PCR 96 well plates (Applied Biosystems, Foster City CA) and ampli cation was conducted in a 7500 Fast Real-Time PCR System (Applied Biosystem) using the following conditions: 50°C for 15 min, 95°C for 5 min, then 45 cycles of 95° C 3 sec and 55°C for 45 sec. All clinical samples and controls were tested in triplicate.
dRT-PCR and RT-PCR from clinical samples.
We evaluated dRT-PCR ampli cation in vLAB and for some experiments we compared dRT-PCR vs standard RT-PCR for SARS-CoV-2. For direct ampli cation, we used RT-PCR conditions, reagents and clinical samples diluted as described before. For standard ampli cation we puri ed SARS-CoV-2 RNA using QIAamp® Viral RNA Mini Kit (Qiagen, Valencia CA) following vendor protocol provided by WRCEVA. For clinical samples, we used 100µL in VTM treated with Trizol followed by modi ed chloroform separation and RNA isolation using the Qiagen RNeasy Mini Kit (Qiagen). The cDNA synthesis was carried out using the iScript Select cDNA Synthesis Kit (Bio-Rad) following the manufacturer's protocol or the puri ed RNA was stored at -80°C until use. All clinical samples were previously analyzed in the clinical diagnostic laboratory at UTMB. For SARS-CoV-2 detection, the laboratory used the Panther Fusion® System. The Fusion SARS-CoV-2 assay involves the following steps: sample lysis, nucleic acid capture, elution transfer, and multiplex RT-PCR. Nucleic acid capture and elution takes place in a single tube on the Panther Fusion system. The eluate is transferred to the Panther Fusion system reaction tube containing the assay reagents. Multiplex RT-PCR is then performed for the eluted nucleic acid on the Panther Fusion system. The Panther Fusion SARS-CoV-2 assay ampli es and detects two conserved regions of the ORF1ab gene in the same uorescence channel, ORF1ab Region 1 ORF1ab Region 2. For dRT-PCR and RT-PCR experiments conducted in our laboratory, we used PCR conditions described before and 2 µl of VTM diluted in vLAB 10X or eluted RNA (puri ed with QIAGEN columns).

SARS-CoV-2 RNA ampli cation in v-LAB.
IGEPAL-630 has been previously used for direct RT-PCR ampli cation and RNA sequencing (9. 10). Our goal here was to use this reagent to conduct direct ampli cation in suspected COVID-19 samples. However, is not known if IGEPAL-630 affects SARS-CoV-2 RNA integrity or if this detergent inhibits activity of enzymes included in the RT-PCR COVID detection kits approved by CDC. To adress this question, initially we tested ampli cation of viral RNA spiked in a viral Lysis Ampli cation Buffer (vLAB) [0.25% IGEPAL, 150 mM NaCL, Tris 10 mM, BSA 1X] using FDA aproved primers/probes for detection of nucleocapside genes N1 and N2. For these experiments, we used serial dilutions (10-fold) of pure RNA diluted in vLAB or water following CDC protocol. We evaluated RT-PCR performance comparing standard curves of RNA in water vs vLAB. Coe cients of correlation (R 2 ) obtained from standard curves showed identical values for N1 and N2 genes in both samples (Fig. 1) with a limit of detection of 5 copies per reaction, and slope and E value (E=PCR e ciency) also did not show signi cant differences between samples diluted in vLAB and water. These results demonstrated that vLAB buffer does not affect SARS-Cov2 RNA integrity and does not inhibit PCR reagents during ampli cation.

SARS-CoV-2 inactivation in v-LAB.
Clinical samples collected in COVID-19 patients are commonly manipulated in BSL-2 laboratories for diagnostic purposes. Therefore, to avoid the risk of exposure of laboratory workers SARS-CoV-2 must rst be inactivated. Other groups have demonstrated that detergents like sodium-dodecyl-sulfate (SDS) and Triton-X100 added to guanidinium thiocyanate-lysis buffers can reduce virus infectivity. However, inactivation of SARS-CoV-2 with IGEPAL-630 has not previously been determined.
Thus, here we investigated the effect of vLAB on virus replication using a uorescent SARS-CoV-2 strain (stable mNeonGreen) to infect Vero E6 cells. For these experiments, virus was diluted in MEM culture medium (positive control) or vLAB and then samples were incubated 10 and 20 min. After incubation, the samples were used to infect cultured cells (P1).
We analyzed infection on monolayers of Vero E6 cells by uorescent microscopy after 3 days of inoculation. Microscopy analysis showed no infection in groups of cells exposed to virus incubated with vLAB for 10 and 20 minutes; only uorescence in the positive control was observed ( Fig. 2A). To con rm viral inactivation, supernatants obtained from P1 were used to re-infect Vero E6 cells (P2). However, only untreated samples (positive control) showed viral replication and no infection was detected in samples treated with vLAB (Fig. 2B). Overall, cell culture assays demonstrate that vLAB inactivates SARS-CoV-2, therefore, it should be feasible to conduct molecular diagnostics in BSL-2 labs using clinical samples inactivated with vLAB.

Direct ampli cation of SARS-CoV-2.
Our initial results demonstrated feasibility to amplify pure RNA diluted in vLAB. However, infected cells within clinical samples can contain inhibitory molecules that can affect PCR. Therefore, we tested if SARS-CoV-2 from cell lysates can be ampli ed directly by standard RT-PCR (CDC protocol). For these experiments, we diluted 10 µl of Vero E6 cells infected with SARS-CoV-2 (1x10 7 pfu/ml) with 90 µl of vLAB (Pass 1, P1 sample). We incubated the sample for 10 or 20 min at room temperature and then we tested direct ampli cation using as template 2 µl of P1. In addition, we tested lysates from P2 ( Table 1).
As expected, we only had ampli cation in samples from P1 but not in negative samples (uninfected), which demonstrated the feasibility of direct ampli cation (Table 1). In P1 samples, we observed CT values of 31-33 in samples incubated for 10 min and over time the CT values increased by 2 cycles (36-35 for 20 min). These results suggest that samples stored at room temperature are susceptible to degradation over the time. This degradation could be explained due to residual activity of RNAses.
Therefore, physical or chemical inactivation of RNAses must be conducted in clinical samples inactivated with vLAB.

Heated RNA of SARS-Cov-2 is ampli ed by dRT-PCR
We investigated the effect of temperature on dRT-PCR using samples viral RNA diluted in vLAB as template. For these experiments, we incubated RNA spiked in vLAB at 65°C for 10 min and 95°C for 2 min. The results showed a slight variation in PCR e ciency in the samples incubated at room temperature (Fig. 3). However, overall, the limit of detection (5 copies per reaction) and CT values were not affected by high temperature and similar values to ampli cation at room temperature were observed. This result con rmed that vLAB and high temperature does not affect RNA integrity or downstream PCR ampli cation. Thus, it should be feasible to use heat for enzymatic inactivation on infected cells diluted in vLAB.

SARS-CoV-2 is ampli ed in heat inactivated samples.
We investigated the effect of temperature on dRT-PCR using infected cells in vLAB as template. We hypothesized that heat inactivation of RNAses reduces viral RNA degradation in samples inactivated with vLAB, therefore, incubation of samples in vLAB at high temperature should enhance PCR ampli cation. To test this hypothesis, we investigated the effect of high temperature on direct ampli cation using lysates of infected cells in vLAB as template. Samples heated at 65°C x 10 min and 95°C x 2 min showed similar CT values. However, these CT values are lower (Table 2) compared with values previously obtained in samples incubated at room temperature. Thus, ampli cation is more e cient in heated samples. This result con rms that incubation of samples obtained in vLAB at high temperatures enhance ampli cation of SARS-CoV-2 by RT-PCR. Therefore, enzymatic heat inactivation should be an included step to enhance sensitivity of the assay in clinical samples inactivated with vLAB.

SARS-CoV-2 is detected in clinical samples diluted in vLAB.
Since the goal of this preliminary work was to demonstrate the feasibility to conduct direct ampli cation of COVID-19 samples, we next tested direct ampli cation in heat-inactivated clinical samples that were diluted in vLAB. We used 30 nasopharyngeal clinical samples previously tested by RT-PCR at UTMB hospital (15 positive and 15 negative). All nasopharyngeal samples were obtained with standard protocols using swabs and placed in viral transport media. For direct RT-PCR, we inactivated samples using 90 µl of sample and 10 µl of a concentrated solution of 10x vLAB. After dilution, samples were incubated 65°C for 10 min and then placed on ice until use; Two microliters of the lysates were used for RT-PCR detection following the CDC protocol. To evaluate direct RNA ampli cation, we compared CT values from dRT-PCR vs CT values of clinical samples (previously tested in UTMB clinical laboratory), however for dRT-PCR we isolated RNA with the QIAmp DSP Viral RNA Mini Kit (Table 3). Our results showed 100% correlation for detecting positive and negative samples comparing QIAmp extraction vs vLAB, thus, we detected 15 positive and 15 negative samples with both methods. We observed a slight variation on CT values for both methods, however, this variation was mainly observed in samples with low CT values (samples with high-moderate viral load, e.g. sample #27, 16, and 24). Thus, this variation does not affect the qualitative results. In other experiments, we conducted a blinded study to evaluate the performance of dRT-PCR in clinical samples (previously tested at UTMB clinical laboratory) with high and low viral loads (Table 4). In these experiments, the dRT-PCR in vLAB method detected 100% of the positive and negative samples with high loads (>30 CT) and only 1 positive sample with low viral load was not detected since the CT value was out of the limit of detection (CT 40). This result demonstrates the feasibility to use concentrated vLAB for direct ampli cation of SARS-CoV-2 in heat-inactivated samples. Importantly, we realize that validation of this method with a larger number of clinical samples is needed.

Data Availability
Raw data supporting the ndings of this study are available from the corresponding author upon reasonable request.

Discussion
We showed that a viral lysis ampli cation buffer (vLAB) prepared with IGEPAL allows dRT-PCR ampli cation of SARS-CoV-2 using primers and protocols approved by the CDC. The COVID-19 pandemic has resulted in an unprecedented worldwide demand for PCR testing. The huge increase in molecular testing has resulted in shortages of PCR reagents, viral transport media (VTM), and viral RNA extraction kits. This problem is exacerbated mainly in undeveloped countries and remote areas were the supply chain for reagents is ine cient. Our goal in this work was to test inexpensive alternatives for molecular detection of SARS-CoV-2 that could be implemented in these low resource settings.
We considered that dRT-PCR ampli cation could be a low-cost alternative for SARS-CoV-2 detection since this technique does not require RNA extraction kits nor specialized equipment for extraction. Currently, there are several commercial reagents for dRT-PCR, however, we decided to test an inexpensive protocol reported by Shatzkes K et al (10). This method is based on the use of a lysis ampli cation buffer prepared with IGEPAL-630 (octylphenoxypolyethoxyethanol). This reagent is a nonionic, non-denaturing detergent that has been used for solubilization, isolation, and puri cation of membrane protein complexes (9,10). Since IGEPAL is a mild detergent that induces cellular lysis but does not inhibit PCR enzymes (9, 10), we hypothesized that vLAB (prepared with IGEPAL) would be optimal for detecting SARS-CoV-2 RNA by dRT-PCR. Our initial studies showed that vLAB components do not inhibit activity of reverse transcriptase and DNA polymerase included in the tested ampli cation kit since we did not observe differences between RNA spiked in vLAB or water (Fig. 1). Our initial studies showed that a vLAB component does not affect activity of reverse transcriptase and DNA polymerase included in the ampli cation kit (Fig. 1). To date, CDC has approved more than 10 RT-PCR kits for COVID detection (https://www.fda.gov/media/134922/download). These kits should also be individually tested for dRT-PCR with vLAB. However, since enzymatic activities and ampli cation reagents included in all approved kits are similar, we anticipate that there will not be signi cant differences observed.
RNA preservation after sample collection is essential to maximize sensitivity and speci city of the detection assay. To prevent degradation, we evaluated two heating temperatures with the objective to inhibit RNAase activity. Both tested temperatures did not affect ampli cation of spiked RNA in the vLAB samples. Recent studies have demonstrated that virus inactivation is achieved at 70°C and here we con rmed heat inactivation in infected cells which were lysed and diluted in vLAB. In addition, we demonstrated the feasibility of conducting dRT-PCR ampli cation in these samples. The vLAB inactivation will allow sample handling in BSL-2 laboratories, thereby reducing the exposure risk of personnel. Clinical samples used in this study were collected in VTM (~3 ml), to enhance viral concentration in the sample, and we used 9 parts of VTM and 1 part of a concentrated solution (10x) of vLAB. Our initial assessment with clinical samples showed a 100% correlation in dRT-PCR with vLAB and RT-PCR conducted with RNA puri ed in our laboratory with a commercial kit, however, we observed variation in CT values that may re ect differences in viral load or sample degradation. To address this question, we conducted additional experiments to evaluate performance of dRT-PCR in samples with differences in viral load. We found that in samples with high viral load (Low CT values <30) we obtained 100% correlation, however, the sensitivity was slightly reduced in samples with high CTs. These results suggest that slight variations in CT values may be due sample degradation during transportation or multiple thawing. A larger experiment with fresh samples should be conducted to validate dRT-PCR results for samples that are close to the limit of detection.
Overall, our results veri ed that using vLAB for molecular diagnostics of SARS-CoV-2 is a feasible method that should be pursued. Since IGEPAL-630 is an inexpensive reagent, the protocol described here could represent an affordable alternative for developing countries or remote areas for molecular detection of SARS-CoV-2.