Introduction:Papillomaviruses are a diverse group of DNA viruses that infect the skin and mucous membranes of humans and a variety of animals. Although more than 100 different human papillomavirus (HPV) types have been characterized HPV6, HPV11, HPV16 and HPV18 are the most clinically relevant. Genital or anal warts (condylomata acuminata or venereal warts) are the most easily recognized sign of genital HPV infection. Although a wide variety of HPV types can cause genital warts, types 6 and 11 accounts for about 90% of all cases. HPV types 16 and 18 are called "high-risk" types because they can cause cervical cancer, as well as anal cancer, vulvar cancer, head and neck cancers, and penile cancer. Other high-risk types include HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 and HPV68. The high-risk types account for over 90% of cervical adenocarcinomas worldwide. HPV-induced cancers often have viral sequences integrated into the cellular DNA. Some of the HPV "early" genes, such as E6 and E7, are known to act as oncogenes that promote tumor growth and malignant transformation
Intended Use:This kit provides a single tube to screen for the presence of high-risk HPV types, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 and HPV68. The multiplex test is detected in two fluorescent channels differentiating between HPV16 / HPV18 which both produce a VIC channel signal and all others which produce a signal in the FAM channel. HPV16 and HPV18 account for 70% of positive findings in clinical practice so it is helpful to know if either of these are present. All other high risk genotypes together make up the remaining 30% of clinical positives and are grouped together into the FAM channel. In this configuration, the kit gives a partial genotyping result and some additional information on which high risk strains are present.
Specificity:The kit contains individual primers directed against each genotype detected in the kit. The primers are designed to exclude detection of ‘low-risk’ and other clinically irrelevant HPV genotypes. The primers and probe sequences in this kit have 100% homology with a broad range of clinically relevant reference sequences based on a comprehensive bioinformatics analysis. They therefore have a very broad quantification profile.
Dynamic Range of Test:Under optimal PCR conditions genesig kits have very high priming efficiencies of >90% and can detect between 1x10e8 and 1x10e2 copies of target template.
Kit Components:1. High risk HPV primer/probe mix (100 reactions BROWN)ROX, FAM, VIC, and Cy5 labelled (see list below)HPV16 ROXHPV18 VICHPV31 FAMHPV33 FAMHPV35 FAMHPV39 FAMHPV45 FAMHPV51 FAMHPV52 FAMHPV56 FAMHPV58 FAMHPV59 FAMHPV66 FAMHPV68 FAMEndogenous control Cy52. High Risk HPV positive control template (RED), Positive signal for ROX, FAM and VIC channelNote: As the HPV16 assay produces amplification through the ROX channel, if using a machine that recommends ROX passive reference dye please ensure ROX normalization is switched off
3. Lyophilized oasig™ Master MixNote: The oasig master mix does not contain ROX, so if using a machine that requires ROX to be used a passive reference dye ensure this is turned off
4. oasig™ resuspension buffer (BLUE)5. RNase/DNase free water (WHITE), for resuspension of primer/probe mix6. Template preparation buffer (YELLOW), for resuspension of positive control template
Reagents and Equipment to be Supplied by the User:Real-Time PCR Instrument (Ability to detect ROX, FAM, VIC and Cy5 channels)DNA extraction kit:This kit is recommended for use with genesig Easy DNA/RNA extraction kit
However, it is designed to work well with all processes that yield high quality DNA with minimal PCR inhibitors
Pipettors and Tips
Vortex and Centrifuge
Thin walled 1.5ml PCR reaction tubes.
Storage and Stability:This kit is stable at room temperature but should be stored at -20ºC on arrival. Once the lyophilized components have been resuspended they should not be exposed to temperatures above -20ºC for longer than 30 minutes at a time and unnecessary repeated freeze/thawing should be avoided. The kit is stable for six months from the date of resuspension under these circumstances.
