INVESTIGADORES
STOLOWICZ Fabiana Gisela
congresos y reuniones científicas
Título:
NEOKIT-COVID19, A COLORIMETRIC REVERSE-TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION TEST TO DETECT SARS-COV-2 AND ITS VARIANTS FROM EXTRACTION-FREE SAMPLES
Autor/es:
WERBAJH SANTIAGO; LAROCCA, LUCIANA; CARRILLO, CAROLINA; STOLOWICZ, FABIANA; OGAS, LORENA; SERGIO PALLOTTO; INÉS ZAPIOLA; SOLANGE CASSARA; MARÍA BELÉN BOUZAS; ADRIÁN VOJNOV
Lugar:
CABA
Reunión:
Congreso; REUNIÓN DE LAS SOCIEDADES DE BIOCIENCIAS 2021; 2021
Institución organizadora:
LXVI REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC) LXIX REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INMUNOLOGÍA (SAI) LIII REUNIÓN ANUAL DE LA ASOCIACIÓN ARGENTINA DE FARMACOLOGÍA EXPERIMENTAL (AAFE) XI REUNIÓN ANUAL DE LA ASOCIACI
Resumen:
Background: The coronavirus disease 2019 caused by SARSCoV-2 has killed millions of people worldwide. Presently, RT-qPCR is considered gold standard test, but requires sophisticated equipment, expertise and is also expensive.Objective: We developed a simple colorimetric molecular test for SARS-CoV-2 based on Reverse Transcription Loop-mediated isothermal Amplification (RT-LAMP). Faster, direct, low-cost and versatile.Methods: we developed the test optimized on nasopharyngeal swab (NP) and saliva samples without an RNA isolation. We optimized of samples pretreatment with lysis buffer and heat-inactivated, temperature, incubation time, enzymes, dNTPs, primers, other components.We used 3 primers set targeting 2 regions of ORF1ab gene and one in ORF E gene. The test result was defined as a Hydroxynaphthol blue dye (HNB) change violet to blue (visible to eye) as a result of the amplification.Results: We present a colorimetric SARS-CoV-2 RNA detection method performed using RT-LAMP to achieve specific, rapid, with a detection limit of 25-100 copies per reaction directly from NP or saliva samples, without RNA isolation. We optimized the final condition of temperature (64°C), time (60m) and sample pretreatment with LB with a heat inactivation of 8 minutes. The assay present 90,6 % of sensitivity and 100 % of specificity.The kit, NEOKIT-PLUS, was authorized by ANMAT, Argentina, after validating it using samples previously analyzed by RT-qPCR. The primer binding sites are well conserved in all the variants of concern (VOC), notified by World Health Organization (WHO). These lineages include B.1.1.7, B.1.351, P.1 and B.1.617.2.