Open AccessOne-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection
Author(s) -
Carmen MezaRobles,
Carlos E Barajas-Saucedo,
Daniel TiburcioJimenez,
Karen A Mokay-Ramírez,
Valery Melnikov,
Iram P. RodríguezSánchez,
Margarita L. MartínezFierro,
Idalia GarzaVeloz,
Sergio A Zaizar-Fregoso,
José Guzmán-Esquivel,
Mario Ramírez-Flores,
Oscar Newton-Sánchez,
Francisco EspinozaGómez,
Osiris G Delgado-Enciso,
Alba S. H. Centeno-Ramirez,
Iván DelgadoEnciso
Publication year - 2020
Publication title -
journal of infection in developing countries
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.322
H-Index - 49
eISSN - 2036-6590
pISSN - 1972-2680
DOI - 10.3855/jidc.12726
Subject(s) - agarose gel electrophoresis , coronavirus , primer (cosmetics) , nucleic acid , biology , loop mediated isothermal amplification , virology , covid-19 , computational biology , genome , agarose , nested polymerase chain reaction , gene , polymerase chain reaction , dna , microbiology and biotechnology , genetics , chemistry , medicine , disease , organic chemistry , pathology , infectious disease (medical specialty)
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.