A novel method for multiplex genotyping in a single reactor using GTPlex-PyroSeq: genotyping HPV as a prototype

Area of Science:

• Molecular Biology
• Genetics
• Virology

Background:

• Accurate identification of human papillomavirus (HPV) genotypes is crucial for disease management.
• Existing methods for HPV genotyping can be limited in detecting multiple infections simultaneously.

Purpose of the Study:

• To introduce and evaluate GTPlex-PyroSeq, a novel multiplex genotyping method for HPV.
• To assess the clinical utility and performance of GTPlex-PyroSeq compared to direct sequencing and HPV DNA chip analysis.

Main Methods:

• GTPlex-PyroSeq utilizes multiplex PCR with type-specific primers containing unique ID sequences, followed by a single pyrosequencing reaction.
• Primers were designed with unique ID sequences to enable genotype-specific nucleotide dispensation during pyrosequencing.
• The assay was validated using HPV plasmids, cell lines, and 80 clinical samples from high-risk HPV-positive patients.

Main Results:

• GTPlex-PyroSeq demonstrated 100% concordance with direct sequencing for single-type HPV infections (70/80 samples).
• The method achieved 97.5% concordance with HPV DNA chip data for single-type infections (50/80 samples).
• GTPlex-PyroSeq outperformed direct sequencing in detecting multiple HPV infections (12/80 samples) with a limit of detection of 100 copies.

Conclusions:

• GTPlex-PyroSeq is a scalable and effective method for multiplex HPV genotyping.
• The assay's open-platform design and adaptability to various sample types suggest broad applicability in clinical settings.
• This novel method offers improved sensitivity for detecting multiple HPV infections.