Multimodal detection of high-risk HPV oncogenic transcripts using a branched hybridization chain reaction amplification method.

The human papillomavirus (HPV) causes most cervical cancer cases. Although cervical cancer is the fourth most common cancer among women globally and has a high mortality rate, it remains the most treatable cancer upon early detection. Compared with HPV DNA tests, HPV mRNA testing is a more effective detection method, as it determines the presence of active oncogenic transcripts. This study presents findings as a proof-of-concept of a multimodal detection biosensor for detecting HPV 16/35 E6 transcripts using a branched hybridization chain reaction (bHCR)-amplification method. Branched HCR hairpins amplified the short RNA targets into long DNA concatemers verified using gel electrophoresis and fluorescence readouts. The bHCR system achieved a lowest detection concentration at 0.125 µM. There was no cross-reactivity with other HPV subtypes (HPV 18, 58, 51, 31, 73, 52, 66, 58, 42, 82, 54, 70 and 44). To further demonstrate the multimodal applicability, bHCR products were evaluated using Cas13a collateral activity and non-enzymatic direct visualization using biotin-labeled hairpins. Lateral flow strips qualitatively confirmed the assay's performance at reducing initiator target concentrations consistent with the fluorescence measurements. Combination of the fluorescence readouts with the complementary visual readouts demonstrates the potential flexibility of the bHCR assay for laboratory and point-of-care settings. The bHCR assay presents a versatile diagnostic strategy for detection of HPV 16/35 oncogenic transcripts while offering a customizable platform for adaptation to other molecular biomarkers.