• Design and optimization of a real-time PCR assay for SARS Coronavirus-2 detection in stool sample
  • Mobin Makhmalbaf,1 Seyed Reza Mohebbi,2,* Seyed Masoud Hosseini,3 Paria Pirasteh,4 Shabnam Kazemian,5 Hamid Asadzadeh-Aghdaei,6
    1. 1 Basic and Molecular Epidemiology of Gastrointestinal Disorders Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran -2. Department of Microbiology and Microbial Biotechnology, Faculty of Li
    2. Research Center for Gastroenterology and Liver Diseases, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
    3. Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran
    4. Research Center for Gastroenterology and Liver Diseases, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
    5. Research Center for Gastroenterology and Liver Diseases, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran


  • Introduction: The coronavirus disease 2019 (COVID-19) outbreak, which was caused by the novel SARS coronavirus 2 (SARS-CoV-2) in Wuhan, China, has spread around the world. Approximately 232 million cases of the disease and over 4.5 million COVID-19-related deaths have been announced worldwide. Due to the presence of SARS-CoV-2 in the COVID-19 patients’ feces, it is really important to detect the viral RNA in stool samples of people suspected of the infection. Because of the high number of patients and the transmission ability of SARS-CoV-2, finding a fast, accurate and at the same time, low-cost assay to diagnose patients with COVID-19 is a major objective, especially in developing countries with limited resources and infrastructure. This study has developed a rapid, simple, and cost-effective diagnostic method based on SYBR green real-time PCR.
  • Methods: The viral RNA genome was extracted from stool samples, and cDNA was synthesized. Bioinformatics software was also used to design and analyze specific primers. The primers targeted a conserved region of the RdRP gene. A real-time polymerase chain reaction was performed and melt curves were analyzed. The results were compared with a commercial detection kit and also PCR and sequencing as a gold standard method.
  • Results: Twenty laboratory positive patient samples and twenty confirmed negative stool samples were used to evaluate the developed method. This assay was also compared to commonly available kits with high sensitivity and specificity. After setting up, the target region was successfully amplified and detected with appropriate efficiency and reproducibility.
  • Conclusion: Given the significant prevalence of SARS-CoV-2 in the feces of COVID-19 patients, it is important to monitor stool samples of suspected people. We developed a method for all SARS-CoV-2 variants based on the results, which can be used as a proper diagnostic test for different SARS-CoV-2 variants.
  • Keywords: SARS-CoV-2 ,COVID-19 ,Real-time PCR, Primer design