RESUMEN
Infectious diseases caused by viruses such as SARS-CoV-2 and HPV have greatly endangered human health. The nucleic acid detection is essential for the early diagnosis of diseases. Here, we propose a method called PLCR (PfAgo coupled with modified Ligase Chain Reaction for nucleic acid detection) which utilizes PfAgo to only use DNA guides longer than 14-mer to specifically cleave DNA and LCR to precisely distinguish single-base mismatch. PLCR can detect DNA or RNA without PCR at attomolar sensitivities, distinguish single base mutation between the genome of wild type SARS-CoV-2 and its mutant spike D614G, effectively distinguish the novel coronavirus from other coronaviruses and finally achieve multiplexed detection in 70 min. Additionally, LCR products can be directly used as DNA guides without additional input guides to simplify primer design. With desirable sensitivity, specificity and simplicity, the method can be extended for detecting other pathogenic microorganisms.
Asunto(s)
Proteínas Argonautas/química , ADN Viral/análisis , Reacción en Cadena de la Ligasa/métodos , Pyrococcus furiosus/enzimología , ARN Viral/análisis , Alphapapillomavirus/química , Alphapapillomavirus/aislamiento & purificación , COVID-19/diagnóstico , ADN Viral/química , Humanos , Límite de Detección , Mutación , Infecciones por Papillomavirus/diagnóstico , ARN Viral/química , SARS-CoV-2/química , SARS-CoV-2/aislamiento & purificación , Sensibilidad y Especificidad , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
In the present study, we upgraded Pyrococcus furiosus Argonaute (PfAgo) mediated nucleic acid detection method and established a highly sensitive and accurate molecular diagnosis platform for the large-scale screening of COVID-19 infection. Briefly, RT-PCR was performed with the viral RNA extracted from nasopharyngeal or oropharyngeal swabs as template to amplify conserved regions in the viral genome. Next, PfAgo, guide DNAs and molecular beacons in appropriate buffer were added to the PCR products, followed by incubating at 95 °C for 20-30 min. Subsequently, the fluorescence signal was detected. This method was named as SARS-CoV-2 PAND. The whole procedure is accomplished in approximately an hour with the using time of the Real-time fluorescence quantitative PCR instrument shortened from >1 h to only 3-5 min per batch in comparison with RT-qPCR, hence the shortage of the expensive Real-time PCR instrument is alleviated. Moreover, this platform was also applied to identify SARS-CoV-2 D614G mutant due to its single-nucleotide specificity. The diagnostic results of clinic samples with SARS-CoV-2 PAND displayed 100% consistence with RT-qPCR test.