One-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection

  • Carmen Meza-Robles Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico
  • Carlos E Barajas-Saucedo Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico
  • Daniel Tiburcio-Jimenez Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico
  • Karen A Mokay-Ramírez Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico
  • Valery Melnikov Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico
  • Iram P Rodriguez-Sanchez Molecular and Structural Physiology Laboratory, School of Biological Sciences, Autonomous University of Nueo León, Monterrey, Nuevo León 64460, Mexico
  • Margarita L Martinez-Fierro Molecular Medicine Laboratory, Academic Unit of Human Medicine and Health Sciences, Autonomous University of Zacatecas, Zacatecas 98160, Mexico
  • Idalia Garza-Veloz Molecular Medicine Laboratory, Academic Unit of Human Medicine and Health Sciences, Autonomous University of Zacatecas, Zacatecas 98160, Mexico
  • Sergio A Zaizar-Fregoso Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico
  • José Guzman-Esquivel Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico
  • Mario Ramirez-Flores Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico
  • Oscar A Newton-Sanchez Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico
  • Francisco Espinoza-Gómez Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico
  • Osiris G Delgado-Enciso Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico
  • Alba SH Centeno-Ramirez Public Health Laboratory, Zacatecas State Health Services, Zacatecas 98600, Mexico
  • Ivan Delgado-Enciso Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico
Keywords: SARS coronavirus, DNA Synthesis, Pandemics, DNA electrophoresis

Abstract

Introduction: Due to the coronavirus pandemic, identifying the infected individuals has become key to limiting its spread. Virus nucleic acid real-time RT-PCR testing has become the current standard diagnostic method but high demand could lead to shortages. Therefore, we propose a detection strategy using a one-step nested RT-PCR.

Methodology: The nucleotide region in the ORF1ab gene that has the greatest differences between the human coronavirus and the bat coronavirus was selected. Primers were designed after that sequence. All diagnostic primers are species-specific since the 3´ end of the sequence differs from that of other species. A primer set also creates a synthetic positive control. Amplified products were seen in a 2.5% agarose gel, as well as in an SYBR Green-Based Real-Time RT-PCR.

Results: Amplification was achieved for the positive control and specific regions in both techniques.

Conclusions: This new technique is flexible and easy to implement. It does not require a real-time thermocycler and can be interpreted in agarose gels, as well as adapted to quantify the viral genome. It has the advantage that if the coronavirus mutates in one of the key amplification nucleotides, at least one pair can still amplify, thanks to the four diagnostic primers.

Published
2020-07-31
How to Cite
1.
Meza-Robles C, Barajas-Saucedo CE, Tiburcio-Jimenez D, Mokay-Ramírez KA, Melnikov V, Rodriguez-Sanchez IP, Martinez-Fierro ML, Garza-Veloz I, Zaizar-Fregoso SA, Guzman-Esquivel J, Ramirez-Flores M, Newton-Sanchez OA, Espinoza-Gómez F, Delgado-Enciso OG, Centeno-Ramirez AS, Delgado-Enciso I (2020) One-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection. J Infect Dev Ctries 14:679-684. doi: 10.3855/jidc.12726
Section
Coronavirus Pandemic