Farzane Zare Ashrafi
1 
, Marzieh Mohseni
1,2 , Maryam Beheshtian
1,2, Zohreh Fattahi
1,2, Fatemeh Ghodratpour
1, Fatemeh Keshavarzi
1, Hanieh Behravan
1, Marzieh Kalhor
1, Khadijeh Jalalvand
1, Maryam Azad
2, Mahdieh Koshki
2, Ali Jafarpour
3,4, Azam Ghaziasadi
3, Alireza Abdollahi
5, Seyed Jalal Kiani
6, Angila Ataei-Pirkooh
6, Iman Rezaei Azhar
3, Farah Bokharaei-Salim
6, Mohammad Reza Haghshenas
7, Farhang Babamahmoodi
7, Zakiye Mokhames
8, Alireza Soleimani
9, Masood Ziaee
10, Davod Javanmard
10, Shokouh Ghafari
10, Akram Ezani
11, Alireza Ansari Moghaddam
12, Fariba Shahraki-Sanavi
12, Seyed Mohammad Hashemi Shahri
13, Azarakhsh Azaran
14, Farid Yousefi
15, Afagh Moattari
16, Mohsen Moghadami
17, Hamed Fakhim
18, Behrooz Ataei
18, Elahe Nasri
18, Vahdat Poortahmasebi
19, Mojtaba Varshochi
20, Ali Mojtahedi
21, Farid Jalilian
22, Mohammad Khazeni
23, Abdolvahab Moradi
24, Alijan Tabarraei
24, Ahmad Piroozmand
25, Yousef Yahyapour
26, Masoumeh Bayani
26, Amir Aboofazeli
3, Parsa Ghafari
3, Fariba Keramat
27, Mahsa Tavakoli
28, Tahmineh Jalali
28,29, Mohammad Hassan Pouriayevali
28,29, Mostafa Salehi-Vaziri
28,29, Hamid Reza Khorram Khorshid
1, Reza Najafipour
1,30, Reza Malekzadeh
31, Kimia Kahrizi
1, Seyed Mohammad Jazayeri
3, Hossein Najmabadi
1,2* 1 Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran
2 Kariminejad-Najmabadi Pathology & Genetics Center, Tehran, Iran
3 Research Center for Clinical Virology, Tehran University of Medical Sciences, Tehran, Iran
4 Gerash Amir-al-Momenin Medical and Educational Center, Gerash University of Medical Sciences, Gerash, Iran
5 Department of Pathology, School of Medicine, Imam Khomeini Hospital, Tehran University of Medical Sciences, Iran
6 Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
7 Department of Medical Microbiology, Antimicrobial Resistance Research Center, Communicable Diseases Institute, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
8 Department of Molecular Diagnostic, Emam Ali Educational and Therapeutic Center, Alborz University of Medical Sciences, Karaj, Iran
9 Department of Infectious Diseases, Imam Ali hospital, Alborz University of Medical Sciences, Karaj, Iran
10 Infectious Diseases Research Center, Birjand University of Medical Sciences, Birjand, Iran
11 Qazvin Deputy of Treatment Reference Laboratory, Qazvin University of Medical Sciences, Qazvin, Iran
12 Health Promotion Research Center, Zahedan University of Medical Science, Zahedan, Iran
13 Infection Disease and Tropical Medicine Research Center, Zahedan University of Medical Science, Zahedan, Iran
14 Department of Medical Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
15 Department of Infectious Diseases, School of Medicine, Razi Hospital, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
16 Department of Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
17 Health policy research center, Shiraz University of medical sciences, Shiraz, Iran
18 Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
19 Department of Bacteriology and Virology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
20 Infectious and Tropical Disease Research Center, Tabriz University of Medical Science, Tabriz, Iran
21 Microbiology Department, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran
22 Department of Medical Virology, Faculty of Medicine, Hamadan University of Medical sciences, Hamadan, Iran
23 Booali lab, Molecular & Virology Diagnostic Section, Qom, Iran
24 Golestan University of Medical Sciences, Gorgan, Iran
25 Department of Microbiology, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran
26 Infectious Diseases and Tropical Medicine Research Center, Babol University of Medical Sciences, Babol, Iran
27 Brucellosis Research Center, Hamedan University of Medical Science, Hamadan, Iran
28 COVID-19 National Reference Laboratory, Pasteur Institute of Iran, Tehran, Iran
29 Department of Arboviruses and Viral Hemorrhagic Fevers (National Reference Laboratory), Pasteur Institute of Iran, Tehran, Iran
30 Cell and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran
31 Digestive Disease Research Institute, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
Abstract
Background: Global real-time monitoring of SARS-CoV-2 variants is crucial to controlling the COVID-19 outbreak. The purpose of this study was to set up a Sanger-based platform for massive SARS-CoV-2 variant tracking in laboratories in low-resource settings.
Methods: We used nested RT-PCR assay, Sanger sequencing and lineage assignment for 930-bp of the SARS-CoV-2 spike gene, which harbors specific variants of concern (VOCs) mutations. We set up our platform by comparing its results with whole genome sequencing (WGS) data on 137 SARS-CoV-2 positive samples. Then, we applied it on 1028 samples from March-September 2021.
Results: In total, 125 out of 137 samples showed 91.24% concordance in mutation detection. In lineage assignment, 123 out of 137 samples demonstrated 89.78% concordance, 65 of which were assigned as VOCs and showed 100% concordance. Of 1028 samples screened by our in-house method, 78 distinct mutations were detected. The most common mutations were: S:D614G (21.91%), S:P681R (12.19%), S:L452R (12.15%), S:T478K (12.15%), S:N501Y (8.91%), S:A570D (8.89%), S:P681H (8.89%), S:T716I (8.74%), S:L699I (3.50%) and S:S477N (0.28%). Of 1028 samples, 980 were attributed as VOCs, which include the Delta (B.1.617.2) and Alpha (B.1.1.7) variants.
Conclusion: Our proposed in-house Sanger-based assay for SARS-CoV-2 lineage assignment is an accessible strategy in countries with poor infrastructure facilities. It can be applied in the rapid tracking of SARS-CoV-2 VOCs in the SARS-CoV-2 pandemic.