Comparison five primer sets from different genome region of COVID-19 for detection of virus infection by conventional RT-PCR

  • Hamid Reza Mollaei Tropical and Infectious Diseases Research Center, Kerman University of Medical Sciences, Kerman, Iran
  • Abass Aghaei Afshar Tropical and Infectious Diseases Research Center, Kerman University of Medical Sciences, Kerman, Iran
  • Davood Kalantar-Neyestanaki Medical Mycology and Bacteriology Research Center, Kerman University of Medical Sciences, Kerman, Iran
  • Mehdi Fazlalipour Department of Arbovirus and Viral Hemorrhagic Fevers (National Ref Lab), Pasteur Institute of Iran (IPI), Tehran, Iran
  • Behnaz Aflatoonian Tropical and Infectious Diseases Research Center, Kerman University of Medical Sciences, Kerman, Iran
Sars-CoV-2; COVID-19; Coronavirus; Reverse transcription-polymerase chain reaction; Specific primer


Background and Objectives: The new beta-coronavirus, which caused Severe Acute Respiratory Coronavirus-2 Syndrome (SARS-CoV-2), a major respiratory outbreak in Wuhan, China in December 2019, is now prevalent in many countries around the world. Identifying PCR-based viruses is a well-known and relatively stable protocol. Unfortunately, the high mutation rates may lead to widespread changes in viral nucleic acid sequences, and so using specific primers for PCR can be recommended. In this study, we evaluated the power of a conventional RT-PCR to detect SARS-CoV-2 RNA among the five set primer sets.
Materials and Methods: The five genomic regions of the Coronavirus SARS-2 virus including Nucleocapsids (N), Envelope (E), RNA depended RNA Polymerase (RdRp), ORF1ab and Spike (S) were selected for primer designing. A conventional RT-PCR was performed to compare sensitivity, specificity and other analytical characteristics of primers designed against two Real Time PCR commercial kits.
Results: The result of the comparative analysis showed that the ORF1ab, N and RdRp primers had a sensitivity, specificity and positive predictive value higher than other primers. A significant difference in the analytical sensitivity between the studied primer sets in RT-PCR kits was observed.
Conclusion: In this study, the ORF1ab, Nucleocapsid and RdRp regions have the best primers for identifying the SARS-CoV-2 RNA between different genes that have been suggested.


1. Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 2020;395:565-574.
2. Madhi A, Ghalyanchilangeroudi A, Soleimani M. Evidence of human coroanvirus (229E), in patients with respiratory infection, Iran, 2015: the first report. Iran J Microbiol 2016;8:316-320.
3. Hu ZB, Ci C. [Screening and management of asymptomatic infection of corona virus disease 2019 (COVID-19)]. Zhonghua Yu Fang Yi Xue Za Zhi 2020;54:E025.
4. Paraskevis D, Kostaki EG, Magiorkinis G, Panayiotakopoulos G, Sourvinos G, Tsiodras S. Full-genome evolutionary analysis of the novel corona virus (2019-nCoV) rejects the hypothesis of emergence as a result of a recent recombination event. Infect Genet Evol 2020;79:104212.
5. Rahman Qureshi UU, Saleem S, Khan A, Afzal MS, Ali MS, Ahmed H. Outbreak of novel Corona virus (2019-nCoV); implications for travelers to Pakistan. Travel Med Infect Dis 2020;101571. doi: 10.1016/j.tmaid.2020.101571.
6. Wu J, Wu X, Zeng W, Guo D, Fang Z, Chen L, et al. Chest CT findings in patients with Corona virus disease 2019 and its relationship with clinical features. Invest Radiol 2020;55:257-261.
7. Xiong Z, Fu L, Zhou H, Liu JK, Wang AM, Huang Y, et al. [Construction and evaluation of a novel diagnosis process for 2019-Corona Virus Disease]. Zhonghua Yi Xue Za Zhi 2020;100:E019.
8. Xu K, Cai H, Shen Y, Ni Q, Chen Y, Hu S, et al. [Management of corona virus disease-19 (COVID-19): the Zhejiang experience]. Zhejiang Da Xue Xue Bao Yi Xue Ban 2020;49:0.
9. Lu R, Zou L, Wang Y, Zhao Y, Zhou W, Wu J, et al. [Sequencing and phylogenetic analyses of structural and accessory proteins of middle east respiratory syndrome Coronavirus from the first imported case in China, 2015]. Bing Du Xue Bao 2015;31:333-340.
10. Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DKW, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 2020;25:2000045.
11. To KK, Tsang OT, Chik-Yan Yip C, Chan KH, Wu TC, Chan JMC, et al. Consistent detection of 2019 novel coronavirus in saliva. Clin Infect Dis 2020;ciaa149. doi: 10.1093/cid/ciaa149.
12. Wang CB. [Analysis of low positive rate of nucleic acid detection method used for diagnosis of novel coronavirus pneumonia]. Zhonghua Yi Xue Za Zhi 2020;100:E010.
13. Won J, Lee S, Park M, Kim TY, Park MG, Choi BY, et al. Development of a laboratory-safe and low-cost detection protocol for SARS-CoV-2 of the Coronavirus disease 2019 (COVID-19). Exp Neurobiol 2020;24:326-333. doi: 10.5607/en20009.
14. Xie C, Jiang L, Huang G, Pu H, Gong B, Lin H, et al. Comparison of different samples for 2019 novel coronavirus detection by nucleic acid amplification tests. Int J Infect Dis 2020;93:264-267.
15. Zhang R, Li JM. [The way to reduce the"false negative results"of 2019 novel coronavirus nucleic acid detection]. Zhonghua Yi Xue Za Zhi 2020;100:E008.
16. Zhang ZW, Zhou YM, Zhang Y, Guo Y, Tao SC, Li Z, et al. Sensitive detection of SARS coronavirus RNA by a novel asymmetric multiplex nested RT-PCR amplification coupled with oligonucleotide microarray hybridization. Methods Mol Med 2005;114:59-78.
17. Yu CH, Liu LT, Liu S, Feng YY, Wang CR, Li HL, et al. [Enhanced-real time PCR: a highly sensitive method for SARS-coronavirus detection]. Beijing Da Xue Xue Bao Yi Xue Ban 2006;38:211-213.
18. Nguyen T, Duong Bang D, Wolff A. 2019 Novel Coronavirus Disease (COVID-19): Paving the Road for Rapid Detection and Point-of-Care Diagnostics. Micromachines (Basel) 2020;11:306.
19. Okba NMA, Widjaja I, Li W, GeurtsvanKessel CH, Farag E, Al-Hajri M, et al. Serologic detection of middle east respiratory syndrome Coronavirus functional antibodies. Emerg Infect Dis 2020;26: 1024-1027.
20. Peiris JS, Poon LL. Detection of SARS coronavirus. Methods Mol Biol 2011;665:369-382.
21. Pfefferle S, Reucher S, Norz D, Lutgehetmann M. Evaluation of a quantitative RT-PCR assay for the detection of the emerging coronavirus SARS-CoV-2 using a high throughput system. Euro Surveill 2020;25:2000152.
22. Spackman E, Kapczynski D, Sellers H. Multiplex real-time reverse transcription-polymerase chain reaction for the detection of three viruses associated with poult enteritis complex: turkey astrovirus, turkey coronavirus, and turkey reovirus. Avian Dis 2005;49:86-91.
23. Stranieri A, Lauzi S, Giordano A, Paltrinieri S. Reverse transcriptase loop-mediated isothermal amplification for the detection of feline coronavirus. J Virol Methods 2017;243:105-108.
24. Tang A, Tong ZD, Wang HL, Dai YX, Li KF, Liu JN, et al. Detection of novel Coronavirus by RT-PCR in stool specimen from asymptomatic child, China. Emerg Infect Dis 2020;26:122-125.
How to Cite
Mollaei HR, Aghaei Afshar A, 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. 12(3):185-193.
Original Article(s)