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Design, Synthesis, and Evaluation of in-house Covid-19 Real-time PCR Diagnostic Kit
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MR Zare1    , Z Ebrahimifar2 , M Sobhani Lari3 , H Shokrollahnia Roshan4 , M Abdollahpour-Alitappeh 5 |
1- Department of Environmental Health Engineering, Larestan University of Medical Sciences, Larestan, Iran,
2- Student Research Committee, Larestan University of Medical Sciences, Larestan, Iran
3- Cellular and Molecular Biology Research Center, Larestan University of Medical Sciences, Larestan, Iran
4- Department of Molecular Virology, Laboratory of Dr Tayibi, Qaimshahr, Iran
5- Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, Iran , abdollahpour1983@yahoo.com |
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Abstract: (162 Views) |
1.Farnoosh G, Alishiri G, Hosseini Zijoud SR, Dorostkar R, Jalali Farahani A. Understanding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease (COVID-19) based on available evidence - a narrative review. Journal of Military Medicine 2020; 22(1): 1-11.
2.Stein RA. The 2019 coronavirus: Learning curves, lessons, and the weakest link. Int J Clin Pract 2020; 74(4): e13488.
3.Murdoch DR, French NP. COVID-19: another infectious disease emerging at the animal-human interface. N Z Med J 2020; 133(1510): 12-5.
4.Yuki K, Fujiogi M, Koutsogiannaki S. COVID-19 pathophysiology: A review. Clin Immunol 2020; 215: 108427.
5.Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020; 395(10223): 497-506.
6.Habas K, Nganwuchu C, Shahzad F, Gopalan R, Haque M, Rahman S, et al. Resolution of coronavirus disease 2019 (COVID-19). Expert Review of anti-Infective Therapy 2020; 18(12): 1201-11.
7.Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A novel coronavirus from patients with pneumonia in china, 2019. The New England Journal of Medicine 2020; 382(8): 727-33.
8.Karim B, Barary M, Fereydouni Z, Sanjari E, Hosseinzadeh R, Salehi-Vaziri M, et al. The nuts and bolts of recombination in the generation of SARS-CoV-2 variants; from XA to XBB. Letters in Applied Microbiology 2024; 77(8); 1-14.
9.Liu YC, Kuo RL, Shih SR. COVID-19: The first documented coronavirus pandemic in history. Biomed J 2020; 43(4): 328-33.
10.Rai P, Kumar BK, Deekshit VK, Karunasagar I, Karunasagar I. Detection technologies and recent developments in the diagnosis of COVID-19 infection. Appl Microbiol Biotechnol 2021; 105(2): 441-55.
11.Stasi C, Fallani S, Voller F, Silvestri C. Treatment for COVID-19: An overview. Eur J Pharmacol 2020; 889: 173644.
12.Yang L, Liu S, Liu J, Zhang Z, Wan X, Huang B, et al. COVID-19: immunopathogenesis and Immunotherapeutics. Signal Transduct Target Ther 2020; 5(1): 128.
13.Magalhaes E, Stoner A, Palmer J, Schranze R, Grandy S, Amin S, et al. An Assessment of Mental Health Outcomes During the COVID-19 Pandemic. Community Ment Health J 2021; 57(7): 1267-77.
14.Maleki A, Mehrbod P, Bokharaei-Salim F, Eybpoosh S, Tavakoli M, Mohammadnejad AE, et al. Epidemiological surveillance of respiratory viral infections in SARS-CoV-2-negative samples during COVID-19 pandemic in Iran. Virol J 2023; 20(1): 296.
15.Worldometer [Available from: https://www.worldometers.info/coronavirus/.
16.Majumder J, Minko T. Recent Developments on Therapeutic and Diagnostic Approaches for COVID-19. AAPS J 2021; 23(1): 14.
17.Ochani R, Asad A, Yasmin F, Shaikh S, Khalid H, Batra S, et al. COVID-19 pandemic: from origins to outcomes. A comprehensive review of viral pathogenesis, clinical manifestations, diagnostic evaluation, and management. Infez Med 2021; 29(1): 20-36.
Sadat Larijani M, Biglari A, Sorouri R, Salehi-Vaziri M, Doroud D, Azadmanesh K, et al. Lessons from COVID-19 Pandemic: A Successful Policy and Practice by Pasteur Institute of Iran. Iran Biomed J 2024; 28(1): 1-7.
19.Saneei D, Jamshidi S, Ghalyanchi Langeroudi A, Akbarein H, Nadji SA, Shoarzargari L, et al. Molecular detection of SARS-CoV-2 in domestic cats in close contact with positively-infected owners in Tehran, Iran in 2021. JFMS Open Rep 2023; 9(2): 20551169231172620.
20.Ahmadi Z, Maleki A, Eybpoosh S, Fereydouni Z, Tavakoli M, Kashanian S, et al. Comparison of a multiplex real-time pcr technique with oxford nanopore technologies next-generation sequencing for identification of SARS-CoV-2 variants of concern. Intervirology 2023; 66(1): 136-41.
21.Yuce M, Filiztekin E, Ozkaya KG. COVID-19 diagnosis -A review of current methods. Biosens Bioelectron 2021; 172: 112752.
22.Espejo AP, Akgun Y, Al Mana AF, Tjendra Y, Millan NC, Gomez-Fernandez C, et al. Review of current advances in serologic testing for COVID-19. Am J Clin Pathol 2020; 154(3): 293-304.
23.Tahamtan A, Ardebili A. Real-time RT-PCR in COVID-19 detection: issues affecting the results. Expert Rev Mol Diagn 2020; 20(5): 453-4.
24.Abbas S, Rafique A, Abbas B, Iqbal R. Real-Time Polymerase chain reaction trends in COVID-19 patients. Pak J Med Sci 2021; 37(1): 180-4.
25.Trujillo AA, McCaustland KA, Zheng DP, Hadley LA, Vaughn G, Adams SM, et al. Use of TaqMan real-time reverse transcription-PCR for rapid detection, quantification, and typing of norovirus. J Clin Microbiol 2006; 44(4): 1405-12.
26.Medhurst AD, Harrison DC, Read SJ, Campbell CA, Robbins MJ, Pangalos MN. The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models. J Neurosci Methods 2000; 98(1): 9-20.
27.Mollaei HR, Afshar AA, Kalantar-Neyestanaki D, Fazlalipour M, Aflatoonian B. Comparison five primer sets from different genome region of COVID-19 for detection of virus infection by conventional RT-PCR. Iran J Microbiol 2020; 12(3): 185-93.
28.Cho H, Jung YH, Cho HB, Kim HT, Kim KS. Positive control synthesis method for COVID-19 diagnosis by one-step real-time RT-PCR. Clin Chim Acta 2020; 511: 149-53.
29.Li D, Zhang J, Li J. Primer design for quantitative real-time PCR for the emerging Coronavirus SARS-CoV-2. Theranostics 2020; 10(16): 7150-62.
30.Falzone L, Gattuso G, Tsatsakis A, Spandidos DA, Libra M. Current and innovative methods for the diagnosis of COVID‑19 infection (Review). Int J Mol Med 2021;47(6): 100.
Armaghane-danesh, Yasuj University of Original Article
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.
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| Keywords: Diagnostic kit, COVID-19, Real-time polymerase |
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Full-Text [PDF 1339 kb]
(41 Downloads)
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Type of Study: Applicable |
Subject:
Microbiology
Received: 2023/11/24 | Accepted: 2024/09/15 | Published: 2024/09/30
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| References |
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1. Farnoosh G, Alishiri G, Hosseini Zijoud SR, Dorostkar R, Jalali Farahani A. Understanding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease (COVID-19) based on available evidence - a narrative review. Journal of Military Medicine 2020; 22(1): 1-11.## 2. Stein RA. The 2019 coronavirus: Learning curves, lessons, and the weakest link. Int J Clin Pract 2020; 74(4): e13488.## [ DOI:10.1111/ijcp.13488] 3. Murdoch DR, French NP. COVID-19: another infectious disease emerging at the animal-human interface. N Z Med J 2020; 133(1510): 12-5.## 4. Yuki K, Fujiogi M, Koutsogiannaki S. COVID-19 pathophysiology: A review. Clin Immunol 2020; 215: 108427.## [ DOI:10.1016/j.clim.2020.108427] 5. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020; 395(10223): 497-506.## [ DOI:10.1016/S0140-6736(20)30183-5] 6. 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Molecular detection of SARS-CoV-2 in domestic cats in close contact with positively-infected owners in Tehran, Iran in 2021. JFMS Open Rep 2023; 9(2): 20551169231172620.## [ DOI:10.1177/20551169231172620] 20. Ahmadi Z, Maleki A, Eybpoosh S, Fereydouni Z, Tavakoli M, Kashanian S, et al. Comparison of a multiplex real-time pcr technique with oxford nanopore technologies next-generation sequencing for identification of SARS-CoV-2 variants of concern. Intervirology 2023; 66(1): 136-41.## [ DOI:10.1159/000534067] 21. Yuce M, Filiztekin E, Ozkaya KG. COVID-19 diagnosis -A review of current methods. Biosens Bioelectron 2021; 172: 112752. [ DOI:10.1016/j.bios.2020.112752] 22. Espejo AP, Akgun Y, Al Mana AF, Tjendra Y, Millan NC, Gomez-Fernandez C, et al. Review of current advances in serologic testing for COVID-19. Am J Clin Pathol 2020; 154(3): 293-304.## [ DOI:10.1093/ajcp/aqaa112] 23. Tahamtan A, Ardebili A. Real-time RT-PCR in COVID-19 detection: issues affecting the results. Expert Rev Mol Diagn 2020; 20(5): 453-4.## [ DOI:10.1080/14737159.2020.1757437] 24. Abbas S, Rafique A, Abbas B, Iqbal R. Real-Time Polymerase chain reaction trends in COVID-19 patients. Pak J Med Sci 2021; 37(1): 180-4.## [ DOI:10.12669/pjms.37.1.3000] 25. Trujillo AA, McCaustland KA, Zheng DP, Hadley LA, Vaughn G, Adams SM, et al. Use of TaqMan real-time reverse transcription-PCR for rapid detection, quantification, and typing of norovirus. J Clin Microbiol 2006; 44(4): 1405-12.## [ DOI:10.1128/JCM.44.4.1405-1412.2006] 26. Medhurst AD, Harrison DC, Read SJ, Campbell CA, Robbins MJ, Pangalos MN. The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models. J Neurosci Methods 2000; 98(1): 9-20.## [ DOI:10.1016/S0165-0270(00)00178-3] 27. Mollaei HR, Afshar AA, Kalantar-Neyestanaki D, Fazlalipour M, Aflatoonian B. Comparison five primer sets from different genome region of COVID-19 for detection of virus infection by conventional RT-PCR. Iran J Microbiol 2020; 12(3): 185-93.## [ DOI:10.18502/ijm.v12i3.3234] 28. Cho H, Jung YH, Cho HB, Kim HT, Kim KS. Positive control synthesis method for COVID-19 diagnosis by one-step real-time RT-PCR. Clin Chim Acta 2020; 511: 149-53.## [ DOI:10.1016/j.cca.2020.10.001] 29. Li D, Zhang J, Li J. Primer design for quantitative real-time PCR for the emerging Coronavirus SARS-CoV-2. Theranostics 2020; 10(16): 7150-62.## [ DOI:10.7150/thno.47649] 30. Falzone L, Gattuso G, Tsatsakis A, Spandidos DA, Libra M. Current and innovative methods for the diagnosis of COVID‑19 infection (Review). Int J Mol Med 2021;47(6): 100.## [ DOI:10.3892/ijmm.2021.4933] |
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Zare M, Ebrahimifar Z, Sobhani Lari M, Shokrollahnia Roshan H, Abdollahpour-Alitappeh M. Design, Synthesis, and Evaluation of in-house Covid-19 Real-time PCR Diagnostic Kit. armaghanj 2024; 29 (4) :497-511 URL: http://armaghanj.yums.ac.ir/article-1-3563-en.html
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