en Infectious diseases and public health Infectious diseases and public health Case Report Case Report <span style="font-size:11pt"><span style="line-height:normal"><span sans-serif="" style="font-family:Calibri,"><span style="font-size:10.0pt"><span new="" roman="" style="font-family:" times="">Precision tracking and monitoring viral genome mutations are critical during a viral pandemic such as COVID-19. As molecular assays for diagnosing numerous infectious agents are being developed, RT-PCR is still deployed as the gold standard for detecting SARS-CoV-2. Despite its proofreading capability, SARS-CoV-2, like other RNA viruses, adopts several changes in its genome. If these mutations, especially deletions, occur in the target areas of primers and probes, they will hinder molecular detection methods from identifying the given gene. The authors describe the cases in which, despite the lack of the <i>N</i> gene detection, the <i>ORF1ab</i> gene was discovered with a relatively low cycle of threshold (Ct). Following sequencing, changes were discovered in the annealing region of the forward and reverse primers and probes used in the SARS-CoV-2 detection kit. Among the most significant mutations is a large deletion of 15 nucleotides in the <i>N</i> gene, which has never been seen in prior variants. This highlights the importance of persistent monitoring of hypervariable regions in the SARS-CoV-2 genome through sequencing and updating the molecular detection kits during the COVID-19 pandemic.</span></span></span></span></span> SARS-CoV-2, COVID-19, N-gene target failure, RT-PCR, Variant 141 145 http://jommid.pasteur.ac.ir/browse.php?a_code=A-10-411-2&slc_lang=en&sid=1 Ali Maleki alimaleki.azt110@gmail.com 0000-0002-6044-3052 No COVID-19 National Reference Laboratory, Pasteur Institute of Iran, Pasteur Ave., 1316943551, Tehran, Iran; 2 Department of Virology, Pasteur Institute of Iran, Pasteur Ave., 1316943551, Tehran, Iran Zahra Fereydouni zahrafereydouni70@gmail.com 0000-0001-8032-6817 No COVID-19 National Reference Laboratory, Pasteur Institute of Iran, Pasteur Ave., 1316943551, Tehran, Iran; 2 Department of Virology, Pasteur Institute of Iran, Pasteur Ave., 1316943551, Tehran, Iran Mahsa Tavakoli mahsa.tavakolirad@gmail.com 0000-0001-8474-5381 No COVID-19 National Reference Laboratory, Pasteur Institute of Iran, Pasteur Ave., 1316943551, Tehran, Iran Akram Ezani akramezani@yahoo.com 0000-0002-3831-4107 No Vice-Chancellor for Treatment Affairs, Reference Laboratory, Qazvin University of Medical Sciences, 34197-59811, Qazvin, Iran Mirshamsedin Hosseini mirshamsedin_1351@yahoo.com 0000-0001-8482-8224 No Vice-Chancellor for Treatment Affairs, Reference Laboratory, Qazvin University of Medical Sciences, 34197-59811, Qazvin, Iran Amir Hesam Nemati hesam.nemati@live.com 0000-0001-7619-5287 No COVID-19 National Reference Laboratory, Pasteur Institute of Iran, Pasteur Ave., 1316943551, Tehran, Iran Parastoo Yektay Sanat p.yekta.oloom@gmail.com 0000-0002-4193-1595 No COVID-19 National Reference Laboratory, Pasteur Institute of Iran, Pasteur Ave., 1316943551, Tehran, Iran Tahmineh Jalali jalalitahmineh@gmail.com 0000-0002-1183-4356 No COVID-19 National Reference Laboratory, Pasteur Institute of Iran, Pasteur Ave., 1316943551, Tehran, Iran; 2 Department of Virology, Pasteur Institute of Iran, Pasteur Ave., 1316943551, Tehran, Iran Mohammad Hassan Pouriayevali m.h.pouria@gmail.com 0000-0003-0133-1517 No Department of Arboviruses and Viral Hemorrhagic Fevers (National Reference Laboratory), Pasteur Institute of Iran, Pasteur Ave., 1316943551, Tehran, Iran Mostafa Salehi-Vaziri mostafavaziri1985@gmail.com 0000-0001-8892-8505 Yes COVID-19 National Reference Laboratory, Pasteur Institute of Iran, Pasteur Ave., 1316943551, Tehran, Iran