Detecção de SARS-COV-2 (COVID-19) através da técnica de amplificação isotérmica mediada por LOOP (LAMP)

Abstract

In 2019, the COVID 19 pandemic caused by the Sars-Cov2-2 virus resulted in millions of cases and deaths worldwide. The diagnosis of the new coronavirus during the COVID-19 pandemic was essential to prevent the spread of the virus and monitor the dynamics of the infection. The main tests for diagnosing COVID-19 include serological and molecular tests. Here, we describe a molecular diagnostic method for the detection of SARS-CoV-2 through the Loop-mediated isothermal amplification (RT-LAMP) technique. We used this technique to detect and amplify genetic sequences of SARSCoV-2 using a set of primers that target regions that encode the N protein. For standardization, we analysed RNA from 858 samples from patients already tested for SARS-CoV -2 by RT-qPCR. The RT-LAMP technique has some obstacles such as false positive and false negative results that can be caused by the acidic pH in the reaction. Therefore, in this study, we evaluated the specificity, sensitivity and accuracy of the RT-LAMP reaction with and without pH adjustment, seeking to standardize the technique to obtain more reliable results. Here we used a total of 868 samples from patients with examination, which were strengths in, in the first analysis, groups according to three syndrome assays performed as the manufacturer, without pH adjustment, and tested an accuracy of 50.96% (k =0.041) compared to RT-qPCR results.This accuracy was improved in the group whose RT-LAMP reaction was pHcorrected to 8-9, with an accuracy of 72.22% (k=0.484). We also observed that the sensitivity increased from 82% (95% CI, 68.56-91.42) to 92.19 (95% CI, 82.70-97.41), the same happening with the specificity, going from 22.22 (95%CI, 12.04 -35.60) to 56.25 (95%CI, 43.28-68.63), after pH adjustment. After standardization with real-time RT-LAMP (rtRT-LAMP), the accuracy was 71.57% (k=0.42), the sensitivity was 77.99% (95% CI, 71.76-83.41), and the specificity improved with 68.35% (95% CI, 63.64-72.79). These data demonstrate that pH adjustment is important to obtain better results in the RT-LAMP technique, contributing to the validation for the development of diagnostics based on LAMP.