Citation: Juthi, R.T.; Sazed, S.A.; Zamil, M.F.; Alam, M.S. Clinical Evaluation of Three Commercial RT-PCR Kits for Routine COVID-19 Diagnosis. Pathogens 2022, 11, 1389. https://doi.org/10.3390/ pathogens11111389 Academic Editor: Jasper Fuk-Woo Chan Received: 24 October 2022 Accepted: 19 November 2022 Published: 21 November 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). pathogens Article Clinical Evaluation of Three Commercial RT-PCR Kits for Routine COVID-19 Diagnosis Rifat Tasnim Juthi † , Saiful Arefeen Sazed † , Md Fahad Zamil and Mohammad Shafiul Alam * International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka 1212, Bangladesh * Correspondence: shafiul@icddrb.org † These authors contributed equally to this work. Abstract: Amongst the multiple ways to diagnose coronavirus disease-2019 (COVID-19), reverse transcription polymerase chain reaction (RT-PCR) remains the reference gold standard, providing fast and accurate results. This study evaluated and compared the performance of three commercially available COVID-19 RT-PCR kits-Aridia ® COVID-19 Real-Time PCR Test (CTK Biotech, Inc., Poway, CA, USA), Novel Coronavirus (2019-nCoV) Nucleic Acid Detection Kit (Sansure Biotech Inc., Chang- sha, China) and Allplex TM 2019-nCoV assay (Seegene Inc., Seoul, Republic of Korea) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A total of 326 clinically suspected patients were enrolled for the study, and among them, 209 were diagnosed as positive and 117 as negative when tested with the reference method, US CDC 2019-Novel Coronavirus (2019-nCoV) Real Time RT-PCR Diagnostic Panel. The Aridia ® kit showed total agreement with the reference test, with a sensitivity of 100% (95% CI: 98.25% to 100.0%) and a specificity of 100% (96.90% to 100.00%). The Allplex TM kit also showed 100% specificity (95% CI: 96.90% to 100.00%), but a lower sensitivity (98.09%, 95% CI: 95.17% to 99.48%). Among the three kits, the Novel Coronavirus (2019-nCoV) Nucleic Acid Detection Kit showed the worst performance, with a sensitivity of 98.6% (95% CI: 95.9% to 99.7%) and a specificity of 95.73, 95% (CI: 90.31% to 98.60%). While all these kits conform to the requirement for routine molecular diagnosis with high performances, the Aridia ® COVID-19 Real-Time PCR Test showed the best performance among the three kits. Keywords: SARS-CoV-2; RT-PCR; COVID-19; real time PCR; diagnostics; RT-PCR kit; real time RT-PCR 1. Introduction COVID-19 has already caused more than 6.5 million deaths and over 625 million cases all over the world (accessed on 21 October 2022) [1]. For fast diagnosis of the disease, there are numerous rapid tests commercially available in the market [2]. Among other diag- nostic methods reverse transcription polymerase chain reaction (RT-PCR), Enzyme-linked Immuno Sorbent Assay (ELISA), radiographic analysis (X-Ray, CT-scan), next-generation sequencing (NGS), and whole genome sequencing (WGS) are currently available [3–6]. Of these, the most common and accurate method to diagnose COVID-19 is quantitative (real-time) RT-PCR analysis of viral RNA [7]. The most used samples for this analysis are nasal, nasopharyngeal, oropharyngeal or naso-oropharyngeal derived, collected with cotton swabs and then transferred into viral transport medium (VTM) [8]. Viral RNA is then extracted from the VTM and subjected to RT-PCR analysis that provides the SARS-CoV-2 status of the sample. There are more than 350 commercially available RT-PCR kits that offer highly sensitive and more specific platforms for the diagnosis of COVID-19 [9]. Several commercially available RT-PCR kits for COVID-19 have obtained authorization for emergency use by various authorities such as World Health Organization (WHO) and the United States food and drug administration (US-FDA). Up to recently, twenty-nine kits have obtained US-FDA Emergency Use Authorization (EUA) [9]. Multiple other kits are also available Pathogens 2022, 11, 1389. https://doi.org/10.3390/pathogens11111389 https://www.mdpi.com/journal/pathogens