High throughput high risk HPV genotyping.
The LMNX Genotyping kit HPV GP HR is an in vitro high-throughput assay based on microsphere suspension technology. reverse hybridization assay using GP5+/6+ primers for the qualitative identification of the individual high risk types HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in DNA extracted from clinical specimens. An interpretation for assigning women at risk for developing cervical cancer can’t be given on the result of this genotyping assay. The kit is used as an aid in the type specific identification of high risk human papillomavirus infections only.
Summary and explanation
Human papillomaviruses (HPV) are small viruses containing a double-stranded, circular DNA genome of approximately 7,900 base pairs. The viral genome contains early (E) and late (L) genes, as well as an untranslated control region . At present, more than 100 different HPV types have been identified based on differences in DNA sequence . HPV types can be subdivided into mucosal types, which can infect anogenital and oral-oropharyngeal mucosa, and cutaneous (skin) HPV types. Among the HPV types infecting the anogenital epithelia, a subset of 14 HPV types have been classified as high-risk [2, 3] for causing alterations of the cervical mucosa and ultimately cervical cancer in women. The LMNX HPV GP HR Genotyping kit allows easy and reliable identification of HPV HR genotypes in PCR products encompassing a highly conserved L1 sequence that is amplified by the GP5+/6+ PCR primers. The GP5+/6+ PCR is a low stringency PCR that also amplifies a fragment from human chromosome 14, which functions as an internal control.
Principle of the procedure
The LMNX kit HPV GP HR consists of 2 kits: the Amplification kit HPV GP and the LMNX HPV GP HR Genotyping kit. The LMNX HPV GP HR Genotyping kit enables easy and reliable identification of 14 high-risk human papillomavirus (HPV) genotypes by hybridization, using xMAP® technology of Luminex® (LMNX).
The Luminex® System is a flexible system for suspension arrays that uses bead-based xMAP® technology. A wide variety of assay types, such as immunoassays, kinase enzyme assays, and interaction assays are performed in an aqueous, homogeneous format, both quickly and efficiently. Multiplexing of assays offers the potential for the simultaneous detection of up to 100 different analytes within a single sample.
With xMAP® technology, molecular reactions take place on the surface of color-coded beads. For each HPV type, target-specific capture probes are covalently linked to a specific set of color-coded beads. Labeled PCR products are captured by the bead-bound capture probes in a hybridization suspension. A microfluidics system delivers the suspension hybridization reaction mixture to a dual laser detection device. A red laser identifies each bead (or HPV probe) by its color-coding, while a green laser detects the hybridization signal associated with each bead (indicating the presence or absence of a particular amplimer).
Table 1. List of beads and coupled HPV genotyping probes
1 CC = conjugate control.
2 IC = internal control.
Identification of HPV genotypes with the LMNX HPV GP HR Genotyping kit is based on the reverse hybridization procedure, using xMAP® technology. Denatured biotinylated amplimers are hybridized with specific oligonucleotide probes, which are immobilized on specific types of beads (Table 1). These amplified products are denatured and hybridized to 14 high risk HPV (HPV-16, 18, -31, -33, -35, 39, -45, -51, -52, -56, -58, -59, -66 and 68) genotype specific probes, also including a variant specific probe of HPV-68. Additionally, a probe directed against the co-amplified fragment of human chromosome 14 is included in the test. The probes are coupled to different microspheres and detected with streptavidin-R-phycoerythrin, which binds to any biotinylated hybrid present. After incubation and additional stringent washing, the samples can be analyzed on the Luminex® System.
REF: K-90 LMNX kit HPV GP HR 96 tests
REF: K-36 EIA kit HPV GP HR 96 tests
REF: K-72 Genotyping kit HPV GP, version 2 20 tests
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2) De Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H. Classification of papillomaviruses. Virol. 324, 17-27 (2004).
3) Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N. Engl. J. Med., 348, 518-527 (2003).
4) Van Doorn LJ, Molijn A, Kleter B, Quint W, Colau B. Highly effective detection of human papillomavirus 16 and 18 DNA by a testing algorithm combining broad-spectrum and type-specific PCR. J Clin Microbiol., 44, 3292-3298 (2006).
5) Van Doorn LJ, Quint W, Kleter B, Molijn A, Colau B, Martin MT, Kravang-In, Torrez-Martinez N, Peyton CL, Wheeler CM. Genotyping of human papillomavirus in liquid cytology cervical specimens by the PGMY line blot assay and the SPF(10) line probe assay. J Clin Microbiol., 40, 979-983 (2002)1) Zur Hausen H. Papillomavirus infections-a major cause of human cancers. Biochim. Biophys. Acta 1288, F55-F78 (1996).