A one-pot method for universal Dengue virus detection by combining RT-RPA amplification and CRISPR/Cas12a assay

Dengue Virus (DENV) is a severe life-threatening virus to human, which is the major cause for dengue fever. However, the efficiency of traditional detection methods unable to meet the additional requirements in clinic practice, including the virus isolation method, ELISA, RT-PCR and qRT-PCR and so on. Therefore, a rapid, simple, and accurate diagnostic for DENV is highly desired. In the current study, we developed a novel method for universal DENV detection via introducing recombinase polymerase amplification (RPA) assay and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and associated (Cas) protein 12a (CRISPR/Cas12a) system in one-pot, achieving the extremely sensitivity and specificity for DENV. The whole detection assay can be finished within 40 min without the requirement for sophisticated equipment. The limit of detection (LoD) was 91.7 copies per test. All the recombinant plasmid of these four serotypes of DENV (I to IV) were successfully identified by using present one-pot DENV universal RT-RPA CRISPR/Cas12a detection system. As for specificity, a total of 22 DENV positive samples were successfully identified, and no-cross reactions were observed in 4 other interferes nuclear acid samples. Moreover, we also established the universal DENV RT-RPA-CRISPR/Cas12a- lateral flow dipstick (LFD) platform and all the four serotypes of DENV (I to IV) were successfully identified, reaching the sensitivity of about 250 copies/test. Together, our present method not only provided an alternative approach for universal DENV detection but also gained a novel insight for other virus identification.