Armaghane Danesh Bimonthly Journal (Jul 2024)
Design, Synthesis, and Evaluation of in-house Covid-19 Real-time PCR Diagnostic Kit
Abstract
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Armaghane-danesh, Yasuj University of Original Article 1056 1056 Medical Sciences Journal (YUMSJ) Design, Synthesis, and Evaluation of in-house Covid-19 Real-time PCR Diagnostic Kit Zare MR1, Ebrahimifar Z2, Sobhani Lari M3, Shokrollahnia Roshan H4, Abdollahpour-Alitappeh M5* 1Department of Environmental Health Engineering, Larestan University of Medical Sciences, Larestan, Iran,2Student Research Committee, Larestan University of Medical Sciences, Larestan, Iran,3Cellular and Molecular Biology Research Center, Larestan University of Medical Sciences, Larestan, Iran,4Department of Molecular Virology, Laboratory of Dr Tayibi, Qaimshahr, Iran,5Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, Iran Received: 24 Nov 2023 Accepted: 15 Sep 2024 Abstract Background & aim: Accurate and sensitive detection of Covid-19 RNA has a highly diagnostic value. Real-time polymerase chain reaction (real-time PCR) is known as the most accurate diagnostic test (a gold standard) used for the diagnosis of Covid-19, having the ability to detect the specific RNA of the virus within a few days of infection. Therefore, the aim of this study was to design a sensitive method based on real-time PCR for the detection of Covid-19. Methods: In the present experimental-applied study, all samplings and laboratory tests were carried out at Imam Reza Hospital, Molecular Laboratory for Corona Diagnosis and Cellular and Molecular Biology Research Center from Larestan University of Medical Sciences, Fars province, Iran, in 2021. Primers and probes of Covid-19 for N and RdRp genes were designed and blasted using the Oligo 7 software and the NCBI website, respectively. After synthesis of the primers and probes, real-time PCR was performed on 100 positive test samples, followed by determination of sensitivity, specificity, stability, precision and validation of the newly-developed kit. The collected data were analyzed using clinical sensitivity and specificity tests. Results: The kit developed in the present study was able to identify all the positive samples of Covid-19. In the sensitivity test, the Limit of Detection (LOD) of the Covid-19 diagnostic kit was found to be 40 copies/mL. By examining the negative and positive samples, the specificity and accuracy of this kit were found to be 100%, and the kit was only able to identify the Covid-19 virus and showed high reproducibility. Temperature shock could not negatively affect the stability and potential of the kit. In addition, there was no significant difference between the kit developed in this study and the non-native commercial kits. Conclusions: Due to its appropriate sensitivity, specificity as well as accuracy and stability compared with similar kits, this kit can be suitable for effective detection of the Covid-19 virus.
