Extraction-free RT-RPA assay for detection of HPV16, HPV18, and HPV45 mRNA expression

Area of Science:

• Molecular diagnostics
• Virology
• Public health

Background:

• High-risk human papillomavirus (hrHPV) mRNA is a more specific biomarker for cervical cancer risk than DNA.
• Current hrHPV mRNA detection methods are complex, expensive, and require specialized equipment, limiting their use in resource-limited settings.

Purpose of the Study:

• To develop a sensitive, specific, and minimally instrumented method for detecting HPV16, HPV18, and HPV45 mRNA in exfoliated cervical cells.
• To create an accessible hrHPV mRNA testing solution for resource-limited settings.

Main Methods:

• Developed real-time reverse transcription recombinase polymerase amplification (RT-RPA) exo assays for HPV16, HPV18, and HPV45 E7 mRNA detection.
• Created an extraction-free sample preparation method using enzymatic lysis and DNA degradation.
• Validated the RT-RPA assays with cultured cells and cervicovaginal samples.

Main Results:

• RT-RPA assays detected ≥100 copies of HPV E7 mRNA per reaction in real time using a portable fluorimeter.
• The extraction-free method allowed specific mRNA amplification from crude cell lysates.
• Testing of 11 cervicovaginal samples showed 100% agreement with RT-qPCR, including positive HPV16 mRNA detection.
• Spiked samples successfully detected HPV16, HPV18, and HPV45 types.

Conclusions:

• The developed RT-RPA method is sensitive and specific for hrHPV mRNA detection.
• The simplified sample preparation and portable detection system offer a viable alternative to current complex methods.
• This prototype assay has the potential to significantly improve access to hrHPV mRNA testing in resource-limited settings, aiding cervical cancer prevention efforts.