1、The Hybrid Capture 2 (hc2) System HPV DNA Test by Digene by Brenda Palacios,Objectives,State the 2 main low-risk HPV DNA types State the 2 main high-risk HPV DNA types State what type of test method is used for the detection of HPV State the minimum sample volume required for testing State what the
2、molecular sandwich consist of,Human Papillomavirus (HPV),Primary etiological agent in cervical cancer 2nd most common type of cancer in women world wide 3rd leading cause of cancer-related deaths in women worldwide Most common viral sexually transmitted infection, that goes undiagnosed due to no sym
3、ptoms developed,HPV Characteristics,Non-enveloped double-stranded DNA virusEpitheliotrophic- has great affinity for epithelial cellsObligatory intracellular parasites that deliver their genome and accessory proteins into host cells for viral replication,HPV Infection,E6 oncogene binds to p53 protein
4、 in host cell p53 protein is a negative regulatorE6 protein mutates p53 protein removing its protective functionMutation disables p53 gene switch, permitting cell to multiply uncontrolled,HPV Types,Types 6 &11most common low-risk HPV Associated with genital warts Rarely found in cervical cancerTypes
5、 16 & 18- most common high-risk HPV Associated with cancers of the cervix, vagina, vulva, anus, and penis,HPV Detection,Traditional screening by Papanicolau (pap smear) testUsed universally for initial detection of intraepithelial abnormalitiesIn case of atypical squamous cells of undetermined signi
6、ficance HPV DNA detection is recommended,www.unsw.edu.au/./sep/cells_image_inside.jpg,Molecular Testing,Hybrid Capture 2 (hc2) System HPV DNA Test Approved by FDANucleic acid hybridization assayNo target DNA amplificationSingle amplification using microplate chemiluminescence for qualitative detecti
7、on of 18 types of HPV, Testing cont.,Differentiates between low and high risk HPV Low-risk HPV: 6, 11, 42, 43, 44 High-risk HPV: 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59, 68Does not determine specific HPV genotype,Controls and Reagents,Controls High-risk control: cloned HPV 16 DNA Low-risk control
8、: cloned HPV 6 DNA Negative control Carrier DNA Calibrators (run in triplicate) Low-risk Calibrator: Cloned HPV 11 DNA High-risk Calibrator: cloned HPV 16 DNA Ensure that the reagents and calibrator materials are functioning properly, for determination of assay cut-off value,Controls and Reagents co
9、nt.,Probes Low-risk Probe: HPV 6, 11, 42, 43, 44 RNA cocktail High-risk Probe: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 58, 59, 68, RNA cocktail Capture Microplate Coated with goat polyclonal anti-RNA:DNA hybrid antibodies Detection Reagent 1 Alkaline phosphatase-conjugated murine monoclonal antibodi
10、es to RNA:DNA hybrids Detection Reagent 2 Chemiluminescent substrate,Specimen Requirements,Cervical specimens collected using a broom type collection device placed in PreservCyt SolutionCervical biopsies btw 2-5 mm in Digene Specimen Transport MediumSpecimens collected with the Digene Cervical Sampl
11、er, placed in SurePath Preservative Fluid4mL of sample needed for denaturation process,Denaturation Process,Samples are mixed with Sample Conversion bufferThen mixed with a 2:1 ratio of Specimen Transport Medium (STM) and Denaturation Reagent (DNR) STM-preservative that retards bacterial growth and
12、retains DNA integrity DNR-dilute sodium hydroxide solution, which lysis cells and denatures HPV DNA,Detection,The Hybrid Capture 2 is a fluid-based molecular hybridization assayDoes not use HPV DNA target amplificationUses hybridized signal amplificationDenatured HPV DNA is hybridized with low-risk
13、or high-risk RNA probeResulting hybrids are captured on microplate wells by immobilized antibody,Detection,Detection is sandwich-style with a second anti-RNA:DNA hybrid conjugated to alkaline-phosphataseBound alkaline-phosphatase is revealed by addition of a chemiluminescent dioxetane-based substrat
14、eSubstrate is cleaved by bound alkaline phosphate and emitted light is measured in a microplate luminometer,www.papillomavirus.cz/images/hybridcapture.jpg,Test Interpretation,Emitted light is measured in Relative Light Units (RLUs) Specimens with RLU/CO ratio 1.7 with low-risk HPV probe are consider
15、ed positive for low-risk HPVSpecimens with RLU/CO ratio 1.7 with high-risk HPV probe are considered positive for high-risk HPVSpecimens with RLU/CO ratio btw 0.80-1.7 are considered indeterminate for either low-risk or high-risk HPV and must be repeated,Limitations,Significant number of false-positi
16、ves (10%-19%) due to cross reactivity with low-risk HPV DNA HPV DNA not amplified Negative predicted value may be compromised in cases in which HPV DNA copy number is low No internal control used for sample sufficiency Not possible to determine if results are due to insufficient DNA or true negative
17、 Large number of inconclusive results Requires large sample volume,Sources of Error,Large concentrations of whole blood, douche, anti-fungal cream, and contraceptive jelly May cause false-negative Contamination of Capture Microplate and Detection Reagent 2 with exogenous alkaline phosphatase May cau
18、se false-positive Presence of nucleases found on human skin and materials Causes nucleic acid degradation In accurate volume delivery of samples and reagents,Conclusion,Due to various types of HPVs the most reliable method of detection is through molecular testingThe Hybrid Capture 2 System helps differentiate btw low-risk and high-risk HPV infectionsIs used in conjunction with pap smears to diagnose, treat, and prevent cervical cancer,
