RESUMO
The outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been challenging human health worldwide. Loop-mediated isothermal amplification (LAMP) has been promptly applied to the detection of SARS-CoV-2 owing to its high amplification efficacy and less requirement of the thermal cycler. However, the vast majority of these LAMP-based assays depend on the non-specific detection of LAMP products, which can not discern the undesirable amplificons, likely to yield unreliable results. Herein, a sequence-specific LAMP assay was reported to detect SARS-CoV-2 using proofreading enzyme-mediated probe cleavage (named Proofman), which could realize real-time and visual detection without uncapping. This assay, introducing a proofreading enzyme and the fluorogenic probe to reverse-transcription LAMP (RT-Proofman-LAMP), can specifically detect the SARS-CoV-2 RNA with a detection limit of 100 copies. In addition to the real-time analysis, the assay is capable of endpoint visualization under a transilluminator within 50 min, providing a convenient reporting manner under the setting of point-of-care testing (POCT). In combination with different fluorophores, the one-pot multiplex assay was successfully achieved to detect multiple targets of SARS-CoV-2 and inner control simultaneously. In summary, the development of RT-Proofman-LAMP offers a versatile and highly-specific method for fast field screening and laboratory testing of SARS-CoV-2, making it a promising platform in COVID-19 diagnosis.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/estatística & dados numéricos , Teste de Ácido Nucleico para COVID-19/estatística & dados numéricos , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Pandemias , Sistemas Automatizados de Assistência Junto ao Leito/estatística & dados numéricos , RNA Viral/análise , RNA Viral/genética , Sensibilidade e EspecificidadeRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can cause acute respiratory distress syndrome, hypercoagulability, hypertension, and multiorgan dysfunction. Effective antivirals with safe clinical profile are urgently needed to improve the overall prognosis. In an analysis of a randomly collected cohort of 124 patients with COVID-19, we found that hypercoagulability as indicated by elevated concentrations of D-dimers was associated with disease severity. By virtual screening of a U.S. FDA approved drug library, we identified an anticoagulation agent dipyridamole (DIP) in silico, which suppressed SARS-CoV-2 replication in vitro. In a proof-of-concept trial involving 31 patients with COVID-19, DIP supplementation was associated with significantly decreased concentrations of D-dimers (P < 0.05), increased lymphocyte and platelet recovery in the circulation, and markedly improved clinical outcomes in comparison to the control patients. In particular, all 8 of the DIP-treated severely ill patients showed remarkable improvement: 7 patients (87.5%) achieved clinical cure and were discharged from the hospitals while the remaining 1 patient (12.5%) was in clinical remission.