A rapid, specific and ultrasensitive detection of the Chikungunya virus based on RT-RPA:CRISPR/Cas12a one-pot dual mode end-point detection system

Abstract

BackgroundChikungunya (CHIK) is an underdiagnosed acute febrile illness (AFI) and an important cause of acute encephalitis syndrome (AES). Unavailaibility of rapid and sensitive molecular point-of-care tests (PoCTs) for CHIK at grass-root level, results in increased hospital burden, due to delayed diagnosis or misdiagnosis with other clinically relevant diseases. Since, no therapeutic intervention is readily available, accurate and differential diagnosis of CHIK is the only available option to initiate early supportive treatment. Thus, we aimed to develop a one-pot reverse transcription recombinase polymerase amplification (RT-RPA) mediated CRISPR/Cas12a based detection platform for rapid, specific, and ultrasensitive detection of chikungunya virus (CHIKV) in clinical samples. ResultsWe have successfully integrated CRISPR/Cas12a technology with reverse transcription recombinase polymerase amplification (RT-RPA) for the detection of Chikungunya virus (CHIKV). The developed assay enabled rapid detection of CHIKV within 35 min, requiring minimal handling process and instrumentation. Next, this assay demonstrated dual mode end-point detection capabilities, employing both fluorescence and lateral flow detection within a reaction. Our one-pot system allows the entire process to be completed without the need to open the reaction tube, thereby eliminating the risk of cross-contamination. Remarkably, the assay exhibits an analytical sensitivity of 412 zg μL−1 (≈1 copy), and 100 % clinical sensitivity and specificity for CHIKV. Furthermore, the developed assay demonstrated limit of detection of 8 gene copies of CHIKV. The assay demonstrates precise detection of CHIKV without any cross-reactivity with other pathogens commonly associated AFI or AES. SignificanceThe overall findings of this study indicate that the RT-RPA:CRISPR/Cas12a detection assay, with one-pot dual-mode detection approach enables rapid, specific and ultrasensitive molecular detection of CHIKV. This advancement holds significant potential for CHIKV detection in resource-limited settings, providing a robust tool for diagnosis and management of the disease. This developed assay may empower clinicians to initiate prompt supportive therapy for Chikungunya fever, thereby improving patient outcomes and public health responses.