key: cord-0934124-7j04co4q authors: Cojocaru, Razvan; Yaseen, Iqra; Unrau, Peter J.; Lowe, Christopher F.; Ritchie, Gordon; Romney, Marc G.; Sin, Don D.; Gill, Sikander; Slyadnev, Maxim title: Microchip RT-PCR Detection of Nasopharyngeal SARS-CoV-2 Samples date: 2021-03-09 journal: J Mol Diagn DOI: 10.1016/j.jmoldx.2021.02.009 sha: 001722468ce3ee15558b7ea17c13dd632268e27a doc_id: 934124 cord_uid: 7j04co4q Fast, accurate and reliable diagnostic tests are critical for controlling the spread of the 2019 Coronavirus disease associated with SARS-CoV-2 infection. The current gold standard for testing is real time PCR (RT-PCR), however, during the current pandemic, supplies of testing kits and reagents have been limited. We report the validation of a rapid (30 min), user-friendly and accurate microchip RT-PCR assay for detection of SARS-CoV-2 from nasopharyngeal swab RNA extracts. Microchips pre-loaded with COVID-19 primers and probes for the N gene accommodate 1.2 μl reaction volumes, lowering the required reagents by 10-fold compared to tube-based RT-PCR. We validated our assay using contrived reference samples and 21 clinical samples from patients in Canada, determining a limit of detection of 1 copy per reaction. The microchip RT-PCR provides a significantly lower resource alternative to the CDC approved real-time RT-PCR assays with comparable sensitivity, showing 100% positive and negative predictive agreement of clinical samples. In late 2019, an outbreak of the novel human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, known as began in Wuhan, Hubei province of China (1) and has rapidly spread around the globe. As of February 26, 2021, over 112.6 million cases and 2,501,229 deaths have been reported worldwide, resulting in enormous economic impact (https://covid19.who.int/). Rapid, sensitive and cost-effective diagnostics can play an important role in the containment of COVID-19 and in bringing society from pandemic to normalcy. Throughout the current pandemic, test kits and reagents required for manufacturing such test kits have been limiting, slowing down the rapid expansion of clinical testing (2) . These limitations have encouraged the search for alternative protocols that use reagents in a more cost effective manner, are userfriendly, and preserve the sensitivity and speed of conventional tests for early-stage detection (3) . Multiple labs around the world are beginning to offer diverse diagnostic solutions, however quantitative reverse transcription polymerase chain reaction (qRT-PCR) remains the current gold standard test used in clinical diagnostics laboratories (4) (5) (6) (7) (8) (9) (10) . The current COVID-19 qRT-PCR test detects the virus by singleplex amplification of one or two segments of either the N, ORF1b, E or RdRp genes, while other assays have been developed to take advantage of multiplex amplification, amplifying multiple genes in a single reaction (4) (5) (6) (7) (8) (9) (10) . Regardless of the method, amidst the pandemic, most of these assays require supply-limited reagents in high volumes and significant technical labor for preparing complex reagent mixtures, resulting in high cost assays with potential for human error (11, 12) . Microchip real-time PCR, an alternative to conventional real-time PCR, has been proven to be a user-friendly technology that can provide reliable, sensitive and specific results in less time (13) (14) (15) (16) (17) (18) . These advantages are attributed to the miniaturized reaction volumes, where the chip's high surface area to volume ratio offers high heat transfer efficiency (13) . This technology is expected to meet the current standards of existing detection assays J o u r n a l P r e -p r o o f (https://www.cdc.gov/flu/professionals/diagnosis/molecular-assays.htm, last accessed January 14, 2021), while giving accurate results in under 30 minutes and addressing the current supply-limiting situations by lowering the cost, labor and reagent consumption. This report presents the clinical validation of a microchip based real time PCR system consisting of a disposable microchip with 30 microreactors (6 columns x 5 rows) where each well can accommodate a miniature TaqMan chemistry-based reaction of 1.2 µl (Figure 1) . The protocol uses a singleplex assay based on 1-step qRT-PCR reactions targeting the SARS-CoV-2-specific N gene. The microchip is preloaded with lyophilized US-CDC recommended N1 and N2 primers and probes for detecting two regions of the N gene, along with HsRPP30 as a human specimen control. The accompanying lightweight AriaDNA microchip-based PCR analyzer, offers user-friendly, rapid real-time monitoring and analysis of PCR reactions while using significantly less resources than tube-based RT-PCR. Therefore, this robust and easy to operate system can be applied for COVID-19 testing in both clinical diagnostics laboratories and point-of-care scenarios. Procurement of reagents. The CDC emergency authorization uses the 2019-nCoV CDC EUA Kit (Cat #10006770) which included primers & probes (Integrated DNA Technology Inc, USA). As per their kit, two primer-probe sets, N1 and N2, were used to detect regions of the N gene of the SARS-CoV-2 virus, and a third primer-probe set to detect HsRPP30, a house keeping gene of human subject as a sample control. UltraPlex 1-Step ToughMix (4X) (Cat # 95166-01K) purchased from Quanta Bio, USA, was utilized as the enzymatic premix for real-time PCR on the microchip. diagnostic test results (19) . In this case, a new sample would have to be collected from the patient and retested, which was not possible due to the wide sample collection timeframe and their subsequent transfer to the university testing laboratory. Further, a blind replication experiment was carried out with the available samples. Randomly, 4 positive and 3 negative patient samples were selected, re-labeled and shuffled by a third-party, to keep sample identity secret to the tester. The tester ran the samples on a microchip with no prior knowledge of the samples, correctly identifying all 7 samples as per previous qRT-PCR assays on the AriaDNA PCR analyzer and conventional qRT-PCR assay (Supplemental Table S5 ). To take advantage of the high heat transfer efficiency the system has to offer, a comparison was performed between the slow thermal cycling settings used in this study (51 min for 45 cycles) and a fast-thermal cycling (30 min for 45 cycles) to potentially speed up testing assays. Four replicates (n = 4) were run each day for 5 consecutive days using 25 copies/µl and 2500 copies/µl of synthetic Armored RNA Quant SARS-CoV-2 RNA. The dCt and CV% data of Ct values obtained for N1, N2 and HsRPP30 targets at slow-and fast-thermal cycling parameters suggest that assays can be performed in as fast as 30 minutes with 45 shorter cycles, satisfying the rapid tests criteria (https://www.cdc.gov/flu/professionals/diagnosis/molecular-assays.htm) (Supplemental Table S6 ). The current golden standard for COVID-19 diagnostic testing is the qRT-PCR assay, a robust technology, but hindered by expensive instrumentation, relatively slow turn-around-times, high cost and low reagent availability, making it restrictive to clinical and public health laboratories amidst the current pandemic. In this report, we validated a microchip qRT-PCR technology for J o u r n a l P r e -p r o o f detection of SARS-CoV-2 in clinical samples. The microchip kit miniaturizes the reaction volumes by 10-fold, resulting in lower reagent consumption and faster assay times (as low as 30 min vs ~70 min), while maintaining the same gold standard in sensitivity as the higher volume techniques. As the kit comes pre-loaded with SARS-CoV-2 primers and probes, it can potentially reduce operatorassociated errors, improving the reliability of analysis in remote settings. The reported assay reliably shows a limit of detection of ~1 viral copy per reaction, with average Ct values that correlate with dilutions ranging from ~1,500 to 1 expected copies per reaction. Notably, the validated assay shows comparable accuracy to that of a clinically validated qRT-PCR assay for the tested 21 COVID-19 nasopharyngeal patient samples. Testing accuracy has been reported to be partially dependent on how the patient samples were collected, with sputum being the most accurate, followed by nasopharyngeal swabs and saliva, and lastly oropharyngeal swabs (23) (24) (25) (26) . Additionally, nasopharyngeal swabs potentially increase the viral exposure of the health care worker in situations where insufficient personal protective equipment is available, increasing the risk of transmission (27) . However, nasopharyngeal samples are currently widely used for diagnostics and have been reported essential for COVID-19 management, including risk assessment of transmission and decision-making regarding quarantine of patients (23) (24) (25) (26) . This study confirms that nasopharyngeal samples can be used to reliably detect SARS-CoV-2 by microchip analysis. As the number of infections and transmissions continues to rise, there is an urgent need for rapid and inexpensive diagnostic tests. Available internationally, the low-energy (100 W), compact, A novel coronavirus outbreak of global health concern. The Lancet SARS-CoV-2 Testing A pneumonia outbreak associated with a new coronavirus of probable bat origin Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP Rapid Detection of Novel Coronavirus (COVID-19) by Reverse Transcription-Loop-Mediated Isothermal Amplification A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry Rapid detection of COVID-19 coronavirus using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform SARS-CoV-2 Detection Using an Isothermal Amplification Reaction and a Rapid, Inexpensive Protocol for Sample Inactivation and Purification RT-qPCR Testing of SARS-CoV-2: A Primer Diagnosing COVID-19: The Disease and Tools for Detection A Novel Multiplex qRT-PCR Assay to Detect SARS-CoV-2 Infection: High Sensitivity and Increased Testing Capacity. Microorganisms COVID-19 diagnostic approaches: different roads to the same destination A fully portable microchip real-time polymerase chain reaction for rapid detection of pathogen Identification and Quantitation of Cashmere (Pashmina) Fiber and Wool Using Novel Microchip Based Real-Time PCR Technology Matrix approach to the simultaneous detection of multiple potato pathogens by real-time PCR Rapid and simple detection of two potato cyst nematode species by real-time multiplex PCR using preserved microarray-based test systems Identification of oncogene mutations in leukemia patients using microchip-based PCR analysis Test-Systems for Monitoring of Corrosion-Relevant Sulfate-Reducting Bacteria Using Real-Time PCR Assay Suboptimal Biological Sampling as a Probable Cause of False-Negative COVID-19 Diagnostic Test Results Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Improved real-time RT-PCR method for highthroughput measurements using second derivative calculation and double correction Comprehensive Algorithm for Quantitative Real-Time Polymerase Chain Reaction Comparative Sensitivity of Different Respiratory Specimen Types for Molecular Diagnosis and Monitoring of SARS-CoV-2 Shedding. The Innovation Nasopharyngeal Swabs Are More Sensitive Than Oropharyngeal Swabs for COVID-19 Diagnosis and Monitoring the SARS-CoV-2 Load Comparison of SARS-CoV-2 detection in nasopharyngeal swab and saliva Saliva or Nasopharyngeal Swab Specimens for Detection of SARS-CoV-2 Swabs Collected by Patients or Health Care Workers for SARS-CoV-2 Testing Acknowledgements: The authors are grateful to Dr. Rajwant Gill, Dr. Irina Gelimson, and Er.Steven Hao for the R&D, QC and technical support concerning the production of lyophilized microchips.