A multiplex recombinase polymerase amplification assay combined with CRISPR/Cas12a for the detection of respiratory syncytial virus and respiratory adenovirus
Gao Hongdan, Yao Du, Qiang Chai, Meng Huang, Liu Xiaorong, Xing Zhihao, Dongli Ma
Abstract
OBJECTIVE: Respiratory syncytial virus (RSV) and respiratory adenovirus (ADV) are two common pathogens that cause acute respiratory tract infections in children. We aimed to develop a rapid method for detecting both pathogens simultaneously. METHODS: The recombinase polymerase isothermal amplification (RPA) method was combined with the CRISPR/Cas detection system. The assay's specificity and sensitivity were explored by designing RPA primers and CRISPR RNAs (crRNAs) through multi-sequence comparisons, optimizing the reaction conditions, and using a fluorescent reading device. The consistency of the test results of 160 clinical pharyngeal swab samples was studied using quantitative polymerase chain reaction (qPCR) results as a comparative control. RESULTS: copies/mL, respectively, within 50 minutes with no cross-reactivity with other similar pathogens. For the clinical samples, compared with the qPCR method, the sensitivities for RSV and ADV were 98.1% and 91.4%, respectively, and the detection specificities were both 100%. The Kappa values were greater than 0.95, suggesting a high degree of consistency. CONCLUSION: This method for detecting RSV and ADV is rapid, sensitive, and specific. It can accurately detect mixed infections in a timely manner, making it suitable for use in areas with scarce healthcare resources.