Carmen Meza-Robles1, Carlos E Barajas-Saucedo2, Daniel Tiburcio-Jimenez3, Karen A Mokay-Ramírez4, Valery Melnikov5, Iram P Rodriguez-Sanchez6, Margarita L Martinez-Fierro7, Idalia Garza-Veloz8, Sergio A Zaizar-Fregoso9, José Guzman-Esquivel10, Mario Ramirez-Flores11, Oscar A Newton-Sanchez12, Francisco Espinoza-Gómez13, Osiris G Delgado-Enciso14, Alba Sh Centeno-Ramirez15, Ivan Delgado-Enciso16. 1. Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico. carmen.qfb@gmail.com. 2. Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico. lalo_cebs@hotmail.com. 3. Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico. dtiburcio@ucol.mx. 4. Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico. ady_mokay@hotmail.com. 5. Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico. valery.melnikoff@gmail.com. 6. Molecular and Structural Physiology Laboratory, School of Biological Sciences, Autonomous University of Nueo León, Monterrey, Nuevo León 64460, Mexico. iramrodriguez@gmail.com. 7. Molecular Medicine Laboratory, Academic Unit of Human Medicine and Health Sciences, Autonomous University of Zacatecas, Zacatecas 98160, Mexico. margarita.mtz.fierro@gmail.com. 8. Molecular Medicine Laboratory, Academic Unit of Human Medicine and Health Sciences, Autonomous University of Zacatecas, Zacatecas 98160, Mexico. 9. Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico. alexzaizar09@gmail.com. 10. Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico. pepeguzman_esquivel@outlook.com. 11. Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico. mario_ramirez@ucol.mx. 12. Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico. onewton@ucol.mx. 13. Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico. fespin@ucol.mx. 14. Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico. 1933osiris@gmail.com. 15. Public Health Laboratory, Zacatecas State Health Services, Zacatecas 98600, Mexico. 16. Department of Molecular Medicine, School of Medicine, University of Colima, Colima 28040, Mexico. ivan_delgado_enciso@ucol.mx.
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. Copyright (c) 2020 Carmen Meza-Robles, Carlos E Barajas-Saucedo, Daniel Tiburcio-Jimenez, Karen A Mokay-Ramirez, Valery Melnikov, Iram P Rodriguez-Sanchez, Margarita L Martinez-Fierro, Idalia Garza-Veloz, Sergio A Zaizar-Fregoso, Jose Guzman-Esquivel, Mario Ramirez-Flores, Oscar A Newton-Sanchez, Francisco Espinoza-Gomez, Osiris G Delgado-Enciso, Alba SH Centeno-Ramirez, Ivan Delgado-Enciso.
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. Copyright (c) 2020 Carmen Meza-Robles, Carlos E Barajas-Saucedo, Daniel Tiburcio-Jimenez, Karen A Mokay-Ramirez, Valery Melnikov, Iram P Rodriguez-Sanchez, Margarita L Martinez-Fierro, Idalia Garza-Veloz, Sergio A Zaizar-Fregoso, Jose Guzman-Esquivel, Mario Ramirez-Flores, Oscar A Newton-Sanchez, Francisco Espinoza-Gomez, Osiris G Delgado-Enciso, Alba SH Centeno-Ramirez, Ivan Delgado-Enciso.
Entities:
Keywords:
DNA Synthesis; DNA electrophoresis; Pandemics; SARS coronavirus
Authors: Ivan Delgado-Enciso; Juan Paz-Garcia; Carlos E Barajas-Saucedo; Karen A Mokay-Ramírez; Carmen Meza-Robles; Rodrigo Lopez-Flores; Marina Delgado-Machuca; Efren Murillo-Zamora; Jose A Toscano-Velazquez; Josuel Delgado-Enciso; Valery Melnikov; Mireya Walle-Guillen; Hector R Galvan-Salazar; Osiris G Delgado-Enciso; Ariana Cabrera-Licona; Eduardo J Danielewicz-Mata; Pablo J Mandujano-Diaz; José Guzman-Esquivel; Daniel A Montes-Galindo; Henry Perez-Martinez; Jesus M Jimenez-Villegaz; Alejandra E Hernandez-Rangel; Patricia Montes-Diaz; Iram P Rodriguez-Sanchez; Margarita L Martinez-Fierro; Idalia Garza-Veloz; Daniel Tiburcio-Jimenez; Sergio A Zaizar-Fregoso; Fidadelfo Gonzalez-Alcaraz; Laydi Gutierrez-Gutierrez; Luciano Diaz-Lopez; Mario Ramirez-Flores; Hannah P Guzman-Solorzano; Gustavo Gaytan-Sandoval; Carlos R Martinez-Perez; Francisco Espinoza-Gómez; Fabián Rojas-Larios; Michael J Hirsch-Meillon; Luz M Baltazar-Rodriguez; Enrique Barrios-Navarro; Vladimir Oviedo-Rodriguez; Martha A Mendoza-Hernandez; Emilio Prieto-Diaz-Chavez; Brenda A Paz-Michel Journal: Exp Ther Med Date: 2021-06-29 Impact factor: 2.447
Authors: Ivan Delgado-Enciso; Juan Paz-Garcia; Carlos E Barajas-Saucedo; Karen A Mokay-Ramírez; Carmen Meza-Robles; Rodrigo Lopez-Flores; Marina Delgado-Machuca; Efren Murillo-Zamora; Jose A Toscano-Velazquez; Josuel Delgado-Enciso; Valery Melnikov; Mireya Walle-Guillen; Hector R Galvan-Salazar; Osiris G Delgado-Enciso; Ariana Cabrera-Licona; José Guzman-Esqu; Daniel A Montes-Galindo; Alejandra E Hernandez-Rangel; Patricia Montes-Diaz; Iram P Rodriguez-Sanchez; Margarita L Martinez-Fierro; Idalia Garza-Veloz; Daniel Tiburcio-Jimenez; Sergio A Zaizar-Fregoso; Mario Ramirez-Flores; Gustavo Gaytan-Sandoval; Carlos R Martinez-Perez; Francisco Espinoza-Gómez; Fabián Rojas-Larios; Michael J Hirsch-Meillon; Enrique Barrios-Navarro; Vladimir Oviedo-Rodriguez; Luz M Baltazar Rodriguez; Brenda A Paz-Michel Journal: Res Sq Date: 2020-09-10