Introducción:
The coronavirus disease 2019 caused by SARS-CoV-2 has killed millions of people worldwide. Presently, RT-qPCR is considered gold standard test, but requires sophisticated equipment, expertise and is also expensive.
Objetivos:
Develop 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.
Materiales y métodos:
We developed an optimized test using nasopharyngeal swab (NP) and saliva samples without an RNA isolation. All the samples used in the present work were remaining diagnostic samples. We optimized sample pretreatment with lysis buffer (LB) 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 visualized as a Hydroxynaphthol blue dye (HNB) change violet to blue (visible to the naked eye) as a result of the amplification.
Resultados:
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 up to the final conditions of temperature (64°C), time (60m). Sample pretreatment includes the adition of LB and a heat inactivation step of 8 minutes. The assay achivied 90,6 % sensitivity and 100 % specificity. The kit was authorized by ANMAT, Argentina, after validating it against 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 Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Epsilon (B.1.427, B.1.429), Delta (B.1.617.2), Omicron (B.1.1.529) and its subtypes.
Discusión / Conclusiones:
We presents a rapid and extraction-free detection of SARS-CoV-2 from NP swab and saliva by colorimetric RT-LAMP. Simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.
