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

摘要: Herein, we describe a novel multiplex genotyping method, GTPlex-PyroSeq. This method consists of two phases: PCR followed by single reaction pyrosequencing. study demonstrates how GTPlex-PyroSeq can be adapted for the determination multiple human papillomavirus (HPV) genotypes. A biotinylated consensus primer, GP6+, and 15 high-risk HPV type-specific primers are used PCR. Each primer has 5′-tag unique ID sequence connected to pyrosequencing binding region. The is composed three parts: i) nucleotide representing specific genotype; ii) sign post; iii) an end mark. design allows genotype under sequence-dependent dispensation order during Following initial studies using plasmids cell lines, evaluated clinical utility effectiveness comparing our assay with direct sequencing DNA chip analysis 80 samples from high-risk, HPV-positive patients. We found in single-type infections, 100% concordance (70 perfect matches) 97.5% data (50 matches). Additionally, system was superior detection infections (12 80), limit 100 copies. scalability this system, its open-platform ability use various sample types, makes GTPlex applicable settings.