RESUMEN
Since SARS-CoV-2 is a highly transmissible virus, a rapid and accurate diagnostic method is necessary to prevent virus spread. We aimed to develop and evaluate a new rapid colorimetric reverse transcription loop--mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection in a single closed tube. Nasopharyngeal and throat swabs collected from at-risk individuals testing for SARS-CoV-2 were used to assess the sensitivity and specificity of a new RT-LAMP assay against a commercial qRT-PCR assay. Total RNA extracts were submitted to the RT-LAMP reaction under optimal conditions and amplified at 65 °C for 30 min using three sets of specific primers targeting the nucleocapsid gene. The reaction was detected using two different indicator dyes, hydroxynaphthol blue (HNB) and cresol red. A total of 82 samples were used for detection with HNB and 94 samples with cresol red, and results were compared with the qRT-PCR assay. The sensitivity of the RT-LAMP-based HNB assay was 92.1% and the specificity was 93.2%. The sensitivity of the RT-LAMP-based cresol red assay was 80.3%, and the specificity was 97%. This colorimetric feature makes this assay highly accessible, low-cost, and user-friendly, which can be deployed for massive scale-up and rapid diagnosis of SARS-CoV-2 infection, particularly in low-resource settings.
RESUMEN
Hepatitis C virus (HCV) infection can be cured with pan-genotypic direct-acting antiviral agents. However, identifying individuals with current hepatitis C remains a major challenge, especially in resource-limited settings where access to or availability of molecular tests is still limited. The goal of this study was to develop and validate a molecular assay for the rapid detection of HCV RNA in resource-limited settings. It is based on a combination of reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 12a (CRISPR-Cas12a) cleavage assay that allows the recognition of specific HCV nucleic acid sequences. Amplified products after the cleavage reactions can be visualized on lateral flow strips or measured with a fluorescence detector. When tested on clinical samples from individuals infected with HCV, HIV, or HBV, or from healthy donors, the RT-LAMP-coupled CRISPR-Cas12 assay yielded 96% sensitivity, 100% specificity, and 97% agreement as compared to the reference method (Roche COBAS AmpliPrep/COBAS TaqMan HCV Test). This assay could detect HCV RNA concentrations as low as 10 ng/µL (an estimated 2.38 Log10 IU/mL). Therefore, this sensitive and specific assay may represent an affordable and reliable point-of-care test for the identification of individuals with active hepatitis C in low-resource settings.
