key: cord-1031938-hcz4tnfg authors: Kiran, Uday; Gokulan, C G; Kuncha, Santosh Kumar; Vedagiri, Dhiviya; Chander, Bingi Thrilok; Sekhar, Aedula Vinaya; Dontamala, Suchitra; Reddy, Arakatla Lohith; Tallapaka, Karthik Bharadwaj; Mishra, Rakesh K; Harshan, Krishnan Harinivas title: Easing diagnosis and pushing the detection limits of SARS-CoV-2 date: 2020-08-20 journal: Biol Methods Protoc DOI: 10.1093/biomethods/bpaa017 sha: 7f6013549419f7ece76bd74ab09767215fb348f8 doc_id: 1031938 cord_uid: hcz4tnfg Rigorous testing is the way forward to fight the COVID-19 pandemic. Here we show that the currently used and most reliable RT-PCR based SARS-CoV-2 procedure can be further simplified to make it faster, safer, and economical by eliminating the RNA isolation step. The modified method is not only fast and convenient but also at par with the traditional method in terms of accuracy, and therefore, can be used for mass screening. Our method takes about half the time and is cheaper by about 40% compared to the currently used method. We also provide a variant of the new method that increases the efficiency of detection by about 30% compared to the existing procedure. Taken together, we demonstrate a more effective and reliable method of SARS-CoV-2 detection. Efficient diagnosis of an infectious pandemic carries inherent challenges such as biosafety during sample handling, skilled manpower, time consumption for testing, sensitivity of the testing method and significant economic burden, irrespective of the nations. The present SARS-CoV-2 is no exception for this, where the most efficient/reliable screening method is Reverse Transcription-Polymerase Chain Reaction (RT-PCR)-based detection of viral nucleic acid from the patient sample [1, 2] . Rapidly growing number of coronavirus disease 2019 (COVID-19) cases warrants reliable and quicker testing methods [3] . In the absence of specific drug and/or vaccine, the only way to control SARS-CoV-2 spread is large scale screening and isolation of the infected individuals at early stages of infection. Screening using antibody-based methods is rapid but cannot be used for early stage detection [4] . Despite being a superior method, RT-PCR demands significant amount of time due to a laborious and expensive RNA isolation step from the Viral Transport Medium (VTM) containing the swab samples. Currently, the challenge is to adapt a detection method which is quicker and still retaining the sensitivity of the standard RT-PCR-based method. Different studies have previously reported inexpensive, nucleic-acid extraction-free methods for PCR-based clinical diagnosis [5, 6] Here we show that the need for VTM as well as RNA isolation step for performing RT-PCR can be completely eliminated by extracting biological samples from dry swabs using TE buffer, which is cost-effective and can be used as a quick screening procedure. In addition, we also show that the sensitivity of the entire RT-PCR based detection is enhanced by about 30%, when using RNA isolated from TE buffer extract compared to the traditional method. The swab samples were collected from voluntary patients at Gandhi Medical College & Hospital, Secunderabad, India. Two nasopharyngeal swabs were collected from each patient and one was transported as dry swab and another in 1 mL VTM (HiMedia Labs, Mumbai) respectively and the samples were kept at 4° C till further processing. Complete sample processing was done in the BSL-3 facility of CSIR-CCMB by following Standard Operating Procedures. a) Resuspension/extraction of biological material from dry swabs: The dry swabs were transferred to 1.5 mL microfuge tubes containing 400 μl of TE buffer [10mM Tris pH-7.4, 0.1mM EDTA 0.1 mM], The swabs were cut to make them fit into the tubes and incubated at room temperature for 30 min to ensure the release of biological material. For direct VTM to RT-PCR, an aliquot of 1 mL VTM samples was diluted 3 times before processing (as the existing recommendation suggests using 3 mL VTM for sample collection). 50 µl of the VTM (for direct VTM to RT-PCR) and TE extract were aliquoted from the respective vials containing swabs in to separate vials and heated at 98° C for 6 min on a dry heat block. The inactivated samples were directly used as a template for RT-PCR. The RNA isolation from 3 mL VTM and TE-buffer (containing dry swab) was performed using the QIAamp Viral RNA isolation kit (Qiagen, Germany) according the manufacturer's protocol. In both cases, 150 μl of the sample was processed for RNA isolation. All the RT-PCR work carried out in a BSL-2 facility of CSIR-CCMB, Hyderabad, India. To further validate the usage of TE buffer extract as a template for direct RT-PCR, we obtained similar samples from twenty-six patients, on the whole taking the sample size to forty. The results have further strengthened our observation that the TE buffer extract is as sensitive as the extracted RNA (n=40; Figure 1B) . Table 3 ). One of the biggest challenges in diagnostics is overcoming the problem of falsenegatives, and SARS-CoV-2 is not an exception to this. Recent reports have shown that the percentage of false negative reported for SARS-CoV-2 is between 20% and 40% with the onset of symptoms and varies with respect to the phase of infection [11, 12] , which is alarming and calls for immediate improvements in the detection methodology. To address this issue, we have combined the TE-extraction method with traditional method that includes RNA-extraction. Here RNA was first isolated from TE extract (described in methodology), followed by RT-PCR. We were pleasantly surprised that almost one-third of the samples (~30%) which were consistently negative with traditional VTM-based method and also direct-RT-PCR method turned out to be positive for SARS-CoV-2. This observation was reproducible in multiple rounds of testing ( Figure 1B, 2A, 2B) . Upon a further closer look at the overall data, it was intriguing to note that the samples which were positive in the TE-based RNA extraction (and negative in other two methods) had a C T value for only one of the two gene (E gene and RdRP), therefore possibly hinting at the low viral load which can be now picked by the new method. The dC T values indicate the increased detection limits of this method as in majority of the samples C T difference between TE-RNA and VTM-RNA was below zero ( Figure 2B ). To rule out any discrepancies in the sample processing we have used RNaseP as an internal control (Supplementary Table 4 ). Interestingly as an indication of RNA amount and quality, the RNase P C T values in case of TE-based approach had lower values compared to RNA isolated from VTM, thereby proving the higher efficiency of the TE-based approach (Supplementary Table 4 ). Therefore, the new hybrid method of TE-based sample extraction results in increasing the overall efficiency by about 30%. These results put forth a remarkable improvement in the detection of SARS-CoV-2 patients with less viral load and therefore provides a better opportunity to manage the pandemic. Further, an improved detection efficiency provides an avenue for adapting this method in combination with pooling strategies. can probably be expanded for screening other respiratory viral infections that are diagnosed using RT-PCR as well [13] . Finally, we also recommend sample collection using dry swab approach which not only eliminates the need of VTM, but also makes the sample handling, transporting, and testing more convenient and safer for the frontline healthcare workers and technicians. Diagnosis of SARS-CoV-2 infection based on CT scan vs RT-PCR: reflecting on experience from MERS-CoV Real-Time Reverse Transcription-Polymerase Chain Reaction Assay for SARS-associated Coronavirus A new coronavirus associated with human respiratory disease in China State of the Science in Dried Blood Spots Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots Heatmapper: web-enabled heat mapping for all DIRECT RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWABS WITHOUT AN RNA EXTRACTION Massive and rapid COVID-19 testing is feasible by extractionfree SARS-CoV-2 RT Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction-Based SARS-CoV-2 Tests by Time Since Exposure Stability issues of RT-PCR testing of SARS-CoV-2 for hospitalized patients clinically diagnosed with COVID-19 Respiratory Viruses Gene Quantification Using Real-Time Quantitative PCR: An Emerging Technology Hits the Mainstream A) Schematic of the entire protocol for TE-based sample extraction and RT-PCR. B) Heatmap representing the C¬T values of E and RdRP genes obtained in two replicates (Rep.1 and Rep.2) of RT-PCR using TE extract, VTM-extracted RNA (VTM-RNA), and TE-extracted RNA (TE-RNA) as templates (n=40) We acknowledge the state government official and the Department of Medical Education, Telangana for providing the samples for this study. UK, CGG and SKK thank the financial support received from UGC, CSIR, and DST (INSPIRE), India, respectively. All the authors acknowledge the support received from CSIR, India. The authors also acknowledge V Devi Prasad (CSIR-CCMB) and Divya Gupta (CSIR-CCMB) for their helpful suggestions.https://mc.manuscriptcentral.com/bmp The authors declare no competing interests