A Novel Method for Multiplex Genotyping in a Single Reactor Using GTPlex-PyroSeq

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