Armaghane Danesh

fa طراحی، ساخت و بررسی کیت تشخیصی واکنش زنجیره‌ای پلیمراز در زمان واقعی(real-time PCR) کوویدـ19 Design, Synthesis, and Evaluation of in-house Covid-19 Real-time PCR Diagnostic Kit میکروبیولوژی Microbiology كاربردي Applicable زمینه و هدف: تشخیص دقیق و حساس RNA کوویدـ19 از ارزش تشخیصی بسیار بالایی برخوردار است. واکنش زنجیره‌ای پلیمراز در زمان واقعی(real-time PCR) دقیق‌ترین تست تشخیصی(استاندارد طلایی) کوویدـ19 شناخته می‌شود که RNA یا ماده ژنتیکی اختصاصی ویروس را ردیابی می‌کند و می‌تواند ویروس را طی چند روز پس از ابتلا به عفونت تشخیص دهد. لذا هدف از این مطالعه, تعیین و طراحی یک روش حساس بر پایه Real-time PCR برای تشخیص کوویدـ19 می‌باشد. روش بررسی: این یک مطالعه تجربی ـ کاربردی میباشد. تمامی نمونه‌گیری‌ها و تست‌های آزمایشگاهی در مرکز آموزشی درمانی امام رضا(ع) لارستان، آزمایشگاه مولکولی تشخیص کرونا دانشکده علوم پزشکی لارستان و مرکز تحقیقات بیولوژی سلولی و مولکولی دانشکده علوم پزشکی لارستان در سال 1400 انجام شد. آغازگرها و کاوشگر اختصاصی کوویدـ19 برای جایگاه ژنی ژن‌های N و RdRp به وسیله نرمافزار الیگو 7 طراحی و سپس با استفاده از سایت NCBI بلاست شد. پس از سنتز آغازگرها و کاوشگرها، کیت real-time PCR بر روی 100 نمونه مثبت امتحان و تست‌&zwj;‌های حساسیت(sensitivity)، اختصاصیت(specificity)، پایداری(stability) و اعتبارسنجی(validation) انجام شد. دادههای جمعآوری شده با استفاده از آزمون حساسیت و اختصاصیت بالینی تجزیه و تحلیل شدند. یافته‌ها: کیت طراحی ‌شده در این مطالعه قادر به شناسایی تمامی نمونه‌های مثبت کوویدـ19 بود. در تست تعیین حساسیت، میزان حد تشخیص (Limit of Detection; LOD) کیت تشخیصی کوویدـ19 تولید شده در این مطالعه 40 کپی در میلی‌لیتر گزارش شد. با بررسی نمونه‌های منفی و مثبت، اختصاصیت و دقت این کیت 100 درصد بوده، فقط قادر به شناسایی ویروس کوویدـ19 بوده و تکرارپذیری بالا را نشان می‌دهد. کیت به‌خوبی شوک دمایی را تحمل کرده و دارای پایداری بالایی می‌باشد. علاوه‌براین، تفاوت معنیداری بین کیت تولید شده در این مطالعه و کیت‌ تجاری خارجی وجود ندارد. نتیجه‌گیری: با توجه به سطح حساسیت، اختصاصیت و همچنین دقت و پایداری مناسب در مقایسه با کیت‌های مشابه، این کیت می‌تواند برای تشخیص مؤثر ویروس کوویدـ19 مناسب باشد.   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: <a href="https://www.worldometers.info/coronavirus/" style="color:blue; text-decoration:underline">https://www.worldometers.info/coronavirus/</a>. 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. <v:rect id="Rectangle_x0020_7" o:gfxdata="UEsDBBQABgAIAAAAIQC2gziS/gAAAOEBAAATAAAAW0NvbnRlbnRfVHlwZXNdLnhtbJSRQU7DMBBF 90jcwfIWJU67QAgl6YK0S0CoHGBkTxKLZGx5TGhvj5O2G0SRWNoz/78nu9wcxkFMGNg6quQqL6RA 0s5Y6ir5vt9lD1JwBDIwOMJKHpHlpr69KfdHjyxSmriSfYz+USnWPY7AufNIadK6MEJMx9ApD/oD OlTrorhX2lFEilmcO2RdNtjC5xDF9pCuTyYBB5bi6bQ4syoJ3g9WQ0ymaiLzg5KdCXlKLjvcW893 SUOqXwnz5DrgnHtJTxOsQfEKIT7DmDSUCaxw7Rqn8787ZsmRM9e2VmPeBN4uqYvTtW7jvijg9N/y JsXecLq0q+WD6m8AAAD//wMAUEsDBBQABgAIAAAAIQA4/SH/1gAAAJQBAAALAAAAX3JlbHMvLnJl bHOkkMFqwzAMhu+DvYPRfXGawxijTi+j0GvpHsDYimMaW0Yy2fr2M4PBMnrbUb/Q94l/f/hMi1qR JVI2sOt6UJgd+ZiDgffL8ekFlFSbvV0oo4EbChzGx4f9GRdb25HMsYhqlCwG5lrLq9biZkxWOiqY 22YiTra2kYMu1l1tQD30/bPm3wwYN0x18gb45AdQl1tp5j/sFB2T0FQ7R0nTNEV3j6o9feQzro1i OWA14Fm+Q8a1a8+Bvu/d/dMb2JY5uiPbhG/ktn4cqGU/er3pcvwCAAD//wMAUEsDBBQABgAIAAAA IQAJu7+ACQIAABYEAAAOAAAAZHJzL2Uyb0RvYy54bWysU9uO2yAQfa/Uf0C8N47TZJu14qxW2aaq tL1I234AwdhGBYYOJM726zvgbDZtX6qqPCCGgcOZM4fVzdEadlAYNLial5MpZ8pJaLTrav71y/bV krMQhWuEAadq/qgCv1m/fLEafKVm0INpFDICcaEafM37GH1VFEH2yoowAa8cJVtAKyKF2BUNioHQ rSlm0+lVMQA2HkGqEGj3bkzydcZvWyXjp7YNKjJTc+IW84x53qW5WK9E1aHwvZYnGuIfWFihHT16 hroTUbA96j+grJYIAdo4kWALaFstVa6Bqimnv1Xz0Auvci0kTvBnmcL/g5UfDw/+Mybqwd+D/BaY g00vXKduEWHolWjouTIJVQw+VOcLKQh0le2GD9BQa8U+Qtbg2KJNgFQdO2apH89Sq2NkkjavykU5 my84k5R7vZwvy9yLQlRPtz2G+E6BZWlRc6RWZnRxuA8xsRHV05HMHoxuttqYHGC32xhkB0Ft3+aR C6AiL48Zx4aaXy9mi4z8Sy78HYTVkfxrtK35cprG6Kgk21vXZHdFoc24JsrGnXRM0iWXhmoHzSPJ iDCakz4TLXrAH5wNZMyah+97gYoz895RK67L+Tw5OQfzxZsZBXiZ2V1mhJMEVfPI2bjcxNH9e4+6 6+mlMtfu4Jba1+qs7DOrE1kyXxb89FGSuy/jfOr5O69/AgAA//8DAFBLAwQUAAYACAAAACEAGH+s H+AAAAAMAQAADwAAAGRycy9kb3ducmV2LnhtbEyPzU7DQAyE70i8w8pI3NpNIpqWkE0FJYgLh1Lg 7m5MErE/UXbbpjw95gS3sWc0/lyuJ2vEkcbQe6cgnScgyGnf9K5V8P72NFuBCBFdg8Y7UnCmAOvq 8qLEovEn90rHXWwFl7hQoIIuxqGQMuiOLIa5H8ix9+lHi5HHsZXNiCcut0ZmSZJLi73jCx0OtOlI f+0OVsEW8XH7/az1Q31+ualp81GTN0pdX033dyAiTfEvDL/4jA4VM+39wTVBGAWzRbrgKIs0W2Ug OHKbLlnseZUvc5BVKf8/Uf0AAAD//wMAUEsBAi0AFAAGAAgAAAAhALaDOJL+AAAA4QEAABMAAAAA AAAAAAAAAAAAAAAAAFtDb250ZW50X1R5cGVzXS54bWxQSwECLQAUAAYACAAAACEAOP0h/9YAAACU AQAACwAAAAAAAAAAAAAAAAAvAQAAX3JlbHMvLnJlbHNQSwECLQAUAAYACAAAACEACbu/gAkCAAAW BAAADgAAAAAAAAAAAAAAAAAuAgAAZHJzL2Uyb0RvYy54bWxQSwECLQAUAAYACAAAACEAGH+sH+AA AAAMAQAADwAAAAAAAAAAAAAAAABjBAAAZHJzL2Rvd25yZXYueG1sUEsFBgAAAAAEAAQA8wAAAHAF AAAAAA== " strokecolor="white" style="position:absolute; margin-left:-34px; margin-top:-85px; width:484.35pt; height:30.3pt; z-index:251668992">Armaghane-danesh, Yasuj University of                                                                                  Original Article <v:shape id="Text_x0020_Box_x0020_5" o:gfxdata="UEsDBBQABgAIAAAAIQC2gziS/gAAAOEBAAATAAAAW0NvbnRlbnRfVHlwZXNdLnhtbJSRQU7DMBBF 90jcwfIWJU67QAgl6YK0S0CoHGBkTxKLZGx5TGhvj5O2G0SRWNoz/78nu9wcxkFMGNg6quQqL6RA 0s5Y6ir5vt9lD1JwBDIwOMJKHpHlpr69KfdHjyxSmriSfYz+USnWPY7AufNIadK6MEJMx9ApD/oD OlTrorhX2lFEilmcO2RdNtjC5xDF9pCuTyYBB5bi6bQ4syoJ3g9WQ0ymaiLzg5KdCXlKLjvcW893 SUOqXwnz5DrgnHtJTxOsQfEKIT7DmDSUCaxw7Rqn8787ZsmRM9e2VmPeBN4uqYvTtW7jvijg9N/y JsXecLq0q+WD6m8AAAD//wMAUEsDBBQABgAIAAAAIQA4/SH/1gAAAJQBAAALAAAAX3JlbHMvLnJl bHOkkMFqwzAMhu+DvYPRfXGawxijTi+j0GvpHsDYimMaW0Yy2fr2M4PBMnrbUb/Q94l/f/hMi1qR JVI2sOt6UJgd+ZiDgffL8ekFlFSbvV0oo4EbChzGx4f9GRdb25HMsYhqlCwG5lrLq9biZkxWOiqY 22YiTra2kYMu1l1tQD30/bPm3wwYN0x18gb45AdQl1tp5j/sFB2T0FQ7R0nTNEV3j6o9feQzro1i OWA14Fm+Q8a1a8+Bvu/d/dMb2JY5uiPbhG/ktn4cqGU/er3pcvwCAAD//wMAUEsDBBQABgAIAAAA IQDobqHTEwIAADIEAAAOAAAAZHJzL2Uyb0RvYy54bWysU9tu2zAMfR+wfxD0vjgJkiw14hRdugwD ugvQ7QNkWbaFyaJGKbGzrx8lp2m2vRXTgyCK0iF5eLi5HTrDjgq9Blvw2WTKmbISKm2bgn//tn+z 5swHYSthwKqCn5Tnt9vXrza9y9UcWjCVQkYg1ue9K3gbgsuzzMtWdcJPwClLzhqwE4FMbLIKRU/o ncnm0+kq6wErhyCV93R7Pzr5NuHXtZLhS117FZgpOOUW0o5pL+OebTcib1C4VstzGuIFWXRCWwp6 gboXQbAD6n+gOi0RPNRhIqHLoK61VKkGqmY2/auax1Y4lWohcry70OT/H6z8fHx0X5GF4R0M1MBU hHcPIH94ZmHXCtuoO0ToWyUqCjyLlGW98/n5a6Ta5z6ClP0nqKjJ4hAgAQ01dpEVqpMROjXgdCFd DYFJulzN16vllFySfIvlW+pqCiHyp98OffigoGPxUHCkpiZ0cXzwIWYj8qcnMZgHo6u9NiYZ2JQ7 g+woSAD7tM7ofzwzlvUFv1nOlyMBL4DodCAlG90VfD2Na9RWpO29rZLOgtBmPFPKxp55jNSNJIah HJiuiIb4N9JaQnUiYhFG4dKg0aEF/MVZT6ItuP95EKg4Mx8tNedmtlhElScjcckZXnvKa4+wkqAK Hjgbj7swTsbBoW5aijTKwcIdNbTWievnrM7pkzBTC85DFJV/badXz6O+/Q0AAP//AwBQSwMEFAAG AAgAAAAhAN3OcADgAAAADgEAAA8AAABkcnMvZG93bnJldi54bWxMj0FPg0AQhe8m/ofNmHgxdhdM iaUsTdNoPLf14m0LUyCys8BuC/XXO5z09t7My5tvss1kW3HFwTeONEQLBQKpcGVDlYbP4/vzKwgf DJWmdYQabuhhk9/fZSYt3Uh7vB5CJbiEfGo01CF0qZS+qNEav3AdEu/ObrAmsB0qWQ5m5HLbylip RFrTEF+oTYe7Govvw8VqcOPbzTrsVfz09WM/dtt+f457rR8fpu0aRMAp/IVhxmd0yJnp5C5UetGy V1HEURbLZZSAmCNqNc9OrJL4JQGZZ/L/G/kvAAAA//8DAFBLAQItABQABgAIAAAAIQC2gziS/gAA AOEBAAATAAAAAAAAAAAAAAAAAAAAAABbQ29udGVudF9UeXBlc10ueG1sUEsBAi0AFAAGAAgAAAAh ADj9If/WAAAAlAEAAAsAAAAAAAAAAAAAAAAALwEAAF9yZWxzLy5yZWxzUEsBAi0AFAAGAAgAAAAh AOhuodMTAgAAMgQAAA4AAAAAAAAAAAAAAAAALgIAAGRycy9lMm9Eb2MueG1sUEsBAi0AFAAGAAgA AAAhAN3OcADgAAAADgEAAA8AAAAAAAAAAAAAAAAAbQQAAGRycy9kb3ducmV2LnhtbFBLBQYAAAAA BAAEAPMAAAB6BQAAAAA= " strokecolor="white" style="position:absolute; margin-left:67px; margin-top:1034px; width:495pt; height:36pt; text-indent:0; z-index:251670016" type="#_x0000_t202"> <v:textbox> <table width="100%"> <tbody> <tr> <td> <div>1056</div> </td> </tr> </tbody> </table> </v:textbox> </v:shape><v:shape id="Text_x0020_Box_x0020_3" o:gfxdata="UEsDBBQABgAIAAAAIQC2gziS/gAAAOEBAAATAAAAW0NvbnRlbnRfVHlwZXNdLnhtbJSRQU7DMBBF 90jcwfIWJU67QAgl6YK0S0CoHGBkTxKLZGx5TGhvj5O2G0SRWNoz/78nu9wcxkFMGNg6quQqL6RA 0s5Y6ir5vt9lD1JwBDIwOMJKHpHlpr69KfdHjyxSmriSfYz+USnWPY7AufNIadK6MEJMx9ApD/oD OlTrorhX2lFEilmcO2RdNtjC5xDF9pCuTyYBB5bi6bQ4syoJ3g9WQ0ymaiLzg5KdCXlKLjvcW893 SUOqXwnz5DrgnHtJTxOsQfEKIT7DmDSUCaxw7Rqn8787ZsmRM9e2VmPeBN4uqYvTtW7jvijg9N/y JsXecLq0q+WD6m8AAAD//wMAUEsDBBQABgAIAAAAIQA4/SH/1gAAAJQBAAALAAAAX3JlbHMvLnJl bHOkkMFqwzAMhu+DvYPRfXGawxijTi+j0GvpHsDYimMaW0Yy2fr2M4PBMnrbUb/Q94l/f/hMi1qR JVI2sOt6UJgd+ZiDgffL8ekFlFSbvV0oo4EbChzGx4f9GRdb25HMsYhqlCwG5lrLq9biZkxWOiqY 22YiTra2kYMu1l1tQD30/bPm3wwYN0x18gb45AdQl1tp5j/sFB2T0FQ7R0nTNEV3j6o9feQzro1i OWA14Fm+Q8a1a8+Bvu/d/dMb2JY5uiPbhG/ktn4cqGU/er3pcvwCAAD//wMAUEsDBBQABgAIAAAA IQBIIFQ/FAIAADIEAAAOAAAAZHJzL2Uyb0RvYy54bWysU9tu2zAMfR+wfxD0vtgJkiw14hRdugwD ugvQ7QMUWY6FyaJGKbGzrx8lu2m2vRXTgyCK0iF5eLi+7VvDTgq9Blvy6STnTFkJlbaHkn//tnuz 4swHYSthwKqSn5Xnt5vXr9adK9QMGjCVQkYg1hedK3kTgiuyzMtGtcJPwClLzhqwFYFMPGQVio7Q W5PN8nyZdYCVQ5DKe7q9H5x8k/DrWsnwpa69CsyUnHILace07+OebdaiOKBwjZZjGuIFWbRCWwp6 gboXQbAj6n+gWi0RPNRhIqHNoK61VKkGqmaa/1XNYyOcSrUQOd5daPL/D1Z+Pj26r8hC/w56amAq wrsHkD88s7BthD2oO0ToGiUqCjyNlGWd88X4NVLtCx9B9t0nqKjJ4hggAfU1tpEVqpMROjXgfCFd 9YFJulzOVstFTi5JvvniLXU1hRDF02+HPnxQ0LJ4KDlSUxO6OD34ELMRxdOTGMyD0dVOG5MMPOy3 BtlJkAB2aY3ofzwzlnUlv1nMFgMBL4BodSAlG92WfJXHNWgr0vbeVklnQWgznCllY0ceI3UDiaHf 90xXJV/Ev5HWPVRnIhZhEC4NGh0awF+cdSTakvufR4GKM/PRUnNupvN5VHkyEpec4bVnf+0RVhJU yQNnw3Ebhsk4OtSHhiINcrBwRw2tdeL6OasxfRJmasE4RFH513Z69Tzqm98AAAD//wMAUEsDBBQA BgAIAAAAIQDdznAA4AAAAA4BAAAPAAAAZHJzL2Rvd25yZXYueG1sTI9BT4NAEIXvJv6HzZh4MXYX TImlLE3TaDy39eJtC1MgsrPAbgv11zuc9PbezMubb7LNZFtxxcE3jjRECwUCqXBlQ5WGz+P78ysI HwyVpnWEGm7oYZPf32UmLd1Ie7weQiW4hHxqNNQhdKmUvqjRGr9wHRLvzm6wJrAdKlkOZuRy28pY qURa0xBfqE2HuxqL78PFanDj28067FX89PVjP3bbfn+Oe60fH6btGkTAKfyFYcZndMiZ6eQuVHrR sldRxFEWy2WUgJgjajXPTqyS+CUBmWfy/xv5LwAAAP//AwBQSwECLQAUAAYACAAAACEAtoM4kv4A AADhAQAAEwAAAAAAAAAAAAAAAAAAAAAAW0NvbnRlbnRfVHlwZXNdLnhtbFBLAQItABQABgAIAAAA IQA4/SH/1gAAAJQBAAALAAAAAAAAAAAAAAAAAC8BAABfcmVscy8ucmVsc1BLAQItABQABgAIAAAA IQBIIFQ/FAIAADIEAAAOAAAAAAAAAAAAAAAAAC4CAABkcnMvZTJvRG9jLnhtbFBLAQItABQABgAI AAAAIQDdznAA4AAAAA4BAAAPAAAAAAAAAAAAAAAAAG4EAABkcnMvZG93bnJldi54bWxQSwUGAAAA AAQABADzAAAAewUAAAAA " strokecolor="white" style="position:absolute; margin-left:67px; margin-top:1034px; width:495pt; height:36pt; text-indent:0; z-index:251667968" type="#_x0000_t202"> <v:textbox> <table width="100%"> <tbody> <tr> <td> <div>1056</div> </td> </tr> </tbody> </table> </v:textbox> </v:shape> Medical Sciences Journal (YUMSJ)</v:rect> 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.   کیت تشخیصی, کوویدـ19, واکنش زنجیره‌ای پلیمراز Diagnostic kit, COVID-19, Real-time polymerase 497 511 http://armaghanj.yums.ac.ir/browse.php?a_code=A-10-2885-1&slc_lang=fa&sid=1 MR Zare محمدرضا زارع 100319475328460030444 100319475328460030444 No Department of Environmental Health Engineering, Larestan University of Medical Sciences, Larestan, Iran, گروه بهداشت محیط، دانشکده علوم پزشکی لارستان، لارستان، ایران Z Ebrahimifar زهرا ابراهیمی‌فر nastaran.ebrahimifar.2002@gmail.com 2283499240 0009-0004-2897-7291 No Student Research Committee, Larestan University of Medical Sciences, Larestan, Iran کمیته تحقیقات دانشجویی، دانشکده علوم پزشکی لارستان، لارستان، ایران M Sobhani Lari محمد سبحانی لاری payamsobhani@yahoo.com 2511302845 0009-0009-2470-8410 No Cellular and Molecular Biology Research Center, Larestan University of Medical Sciences, Larestan, Iran مرکز تحقیقات بیولوژی سلولی و مولکولی، دانشکده علوم پزشکی لارستان، لارستان، ایران H Shokrollahnia Roshan هادی شکرالله‌نیا روشن hadi.roshan1205@gmail.com 2063919720 0009-0000-7068-1313 No Department of Molecular Virology, Laboratory of Dr Tayibi, Qaimshahr, Iran گروه ویروس‌شناسی مولکولی، آزمایشگاه تشخیص طبی دکتر طیبی، قائمشهر، ایران M Abdollahpour-Alitappeh مقداد عبداله‌پور علی‌تپه abdollahpour1983@yahoo.com 5799910494 100319475328460030448 Yes Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, Iran بخش فیزیولوژی و فارماکولوژی، انستیتو پاستور ایران، تهران، ایران