HPV 16 DNA and mRNA in cervical brush samples quantified by PCR and microwell hybridization

Area of Science:

• Molecular Biology
• Oncology
• Virology

Background:

• Human Papillomavirus (HPV) 16 is a major cause of cervical cancer.
• Accurate quantification of HPV 16 DNA and mRNA is crucial for diagnosing and monitoring cervical neoplasia.

Purpose of the Study:

• To develop and validate a quantitative method for HPV 16 DNA and mRNA.
• To investigate the correlation between HPV 16 viral load and cervical neoplasia severity.

Main Methods:

• Developed a colorimetric detection assay using biotinylated amplicons, streptavidin-coated plates, and alkaline phosphatase-labelled probes.
• Quantified HPV 16 DNA and mRNA in 89 cervical samples using PCR and reverse transcription PCR.
• Analyzed viral DNA and mRNA levels in relation to histological/cytological diagnoses.

Main Results:

• Achieved dynamic ranges of 4–6 log10 for HPV 16 DNA and mRNA quantification.
• Demonstrated high reproducibility with coefficients of variation below 8%.
• Found significantly higher HPV 16 DNA copies in CIN II and high-grade neoplasia subgroups compared to CIN I/normal.
• Observed significantly greater HPV 16 mRNA content in the high-grade neoplasia subgroup.
• Identified higher E5 mRNA equivalents per viral DNA copy and higher E6*I mRNA concentrations.

Conclusions:

• The developed colorimetric assay provides a reproducible and sensitive method for quantifying HPV 16 DNA and mRNA.
• Increased HPV 16 viral load and specific mRNA transcripts correlate with the severity of cervical neoplasia.
• Further research may elucidate the role of specific HPV 16 transcripts in cervical carcinogenesis.