Simultaneous Amplification and Identification of 25 Human Papillomavirus Types with Templex Technology

Abstract

The majority of existing human papillomavirus (HPV) genotyping assays are based on multiplex PCR using consensus or degenerate primers. We developed a Templex HPV assay that simultaneously detects and identifies 25 common HPV genotypes in a single-tube reaction using type-specific primers for the HPV-specific E6 and E7 genes. The analytical sensitivities of the Templex assay for HPV type 16 (HPV-16), -18, and -56 were 20, 100, and 20 copies per reaction mixture, respectively. The Templex assay provides semiquantitative information on each type when multiple HPV types coexist in one reaction. We tested 109 clinical cervical specimens previously evaluated with the Digene HC2 high-risk HPV DNA test and found 95.4% concordance between the assay results. The Templex assay provided type-specific results and found multiple types in 29.2% (14 of 48) of high-risk HPV-positive samples. The entire Templex procedure, including DNA extraction, can be completed within 5 hours, providing a rapid and reliable diagnostic tool for HPV detection and typing that is amenable to automation.Human papillomavirus (HPV) infection is linked with cervical cancer (1,12,24). HPV can be divided into "high-risk (HR)" and "low-risk" groups on the basis of their association with cervical lesions (7,11,17). The HR group includes HPV types 16,18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 67, 68, 69, 82, 26, 53, 66, 70, and 73, while the low-risk group includes types 6, 11, 40, 42, 43, 44, 54, 61, 72, 81, and 89 (11, 15, 19). Recently, quadrivalent and bivalent vaccines have been demonstrated to effectively prevent type-specific persistent infection and disease (6, 23). To monitor the impact of vaccine implementation strategies, determine type-specific persistence, and evaluate the clinical significance of coinfection with multiple HPV types, HPV testing will require type-specific results. A high-throughput, sensitive, specific, and reproducible HPV detection and typing assay is therefore highly desirable.Most established HPV typing assays used in epidemiologic studies are based on consensus PCR to amplify the relatively conserved L1 gene region with hybridization, restriction enzyme digestion, or sequencing of the amplicon to determine type(s) (2, 22). Widely used L1 consensus primer PCR systems include the GP5ϩ/6ϩ, PGMY09/11, and SPF systems (4, 5, 13, 21). However, in all of these methods, variations in the efficiency of type-specific priming, primer competition, and limitations on the reagent concentrations in the assay may affect the observed type distribution, particularly when large numbers of types at greatly different copy numbers are present. In addition, typing requires a variable number of additional post-PCR steps, such as amplicon purification, gel electrophoresis, hybridization, and washing. These additional steps increase the time and labor required.We report the development of a novel HPV genotyping assay that is based on proprietary Templex technology. Templex is a unique multiplex PCR platform that uses a targetenriched multiple...

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