RT‐RPA Assisted CRISPR/Cas12a Based One‐Pot Rapid and Visual Detection of the Pan‐Dengue Virus

ABSTRACT Globally ≤ 4 billion of the population are at potential risk of contracting dengue virus (DENV) infection. Seasonal outbreaks of dengue are frequently reported causing a high healthcare burden. Undiagnosed DENV can lead to severe morbidity and mortality. Early diagnosis of DENV relies on molecular methods, which are impractical in resource‐constrained settings (RCSs). Dengue can be caused by any of the four distinct DENV serotypes. Therefore, a simple method for rapid diagnosis of Pan‐DENV serotypes is of utmost importance at RCSs. A fluorescence detection platform for Pan‐DENV using RT‐RPA and CRISPR/Cas12a was developed targeting nonstructural 1 ( NS1 ) gene for DENV‐1, 2, and 3, and envelope ( E ) gene for DENV‐2. Further, crRNA specific to DENV serotypes were designed to facilitate CRISPR/Cas12a detection. Analytical sensitivity was determined using synthetic RNA and DENV serotypes genome. Clinical validation of the assay was performed using RNA extracted from AES/AFI clinical samples. The developed CRISPR/Cas12a‐based detection platform can detect all four serotypes of DENV viz 1−4 in a single pot using fluorescence detection. This assay showed the limit of detection ≥ 781 zg reaction − 1 , ≥ 1.81 ag reaction −1 , ≥ 62.5 fg reaction −1 , and ≥ 2.5 pg reaction −1 for synthetic DENV‐1, DENV‐2, DENV‐3, and DENV‐4 template, respectively. Our assay demonstrated the analytic sensitivity of ≥ 10 ng reaction −1 for DENV‐1 and DENV‐4, and ≥ 0.5 ng reaction −1 for DENV‐3 and DENV‐4 genomes. This assay showed no cross‐reactivity with other related etiologies tested causing AFI/AES. With 76 clinical samples (DENV PCR positive = 16, DENV PCR negative = 60), the assay demonstrated 93.7% sensitivity and 100% specificity with an overall accuracy of 98.7% for detection of the Pan‐DENV serotypes. Our assay displayed comparable results to that of RT‐PCR. The ease of interpretation and rapid detection of the Pan‐DENV, represents the potential of the developed assay as an ideal point‐of‐care test. This assay upon field‐deployment could help in reducing healthcare burden, provide differential diagnosis and support initiating early and prompt treatment to patients at RCS.