Extraction-free RT-qPCR method for the rapid detection of Chikungunya Virus

Abstract

Rapid detection of Chikungunya virus (CHIKV) is critical for effective outbreak management, yet standard RNA extraction protocols are often time-consuming and labour-intensive. This study aimed to optimize an extraction-free RT-qPCR method to facilitate rapid CHIKV detection directly from serum samples. Three extraction-free RT-qPCR methods (direct loading, dilution 1:2, and heat treatment) were screened across a 5-log range (105 – 109 copies/reaction) using purified CHIKV RNA spiked into serum. The optimal protocol was subsequently validated using serum matrices spiked with intact, PEGylated CHIKV (101 – 104 PFU/ml) to simulate clinical specimens. The dilution 1:2 method consistently minimized serum-mediated enzymatic inhibition compared to other methods, achieving lower quantification cycle (Cq) values and strong linearity R 2 of 0.9982 when testing with intact viral particles. The analytical Limit of Detection (LoD) of the optimized 1:2 dilution was of 5 PFU/ml (Cq = 33.19 ± 0.23, 100% replicate positivity), which correspond to 38.4 RNA copies per reaction. The extraction-free methods exhibited lower efficiencies compared to standard extraction-mediated methods, yielding cycle threshold shift ranging from ~5 to 9 cycles. This is expected due to the absence of a chemical concentration step. The typical circulating viremia observed in acute patients exhibit 2.94 x 104 to 1.6 x108 PFU/ml, thus the validated dilution 1:2 extraction-free assay can still serve as a highly viable, economical, and field-deployable alternative to traditional extraction methodologies for rapid CHIKV surveillance.