_papillomavirus detection by the hybrid 混合型人乳头瘤病毒的检测课件

The Hybrid Capture 2 (hc2) The Hybrid Capture 2 (hc2) SystemSystem HPV DNA TestHPV DNA Test byby DigeneDigene by Brenda Palaciosby Brenda Palacios ObjectivesObjectives State the 2 main low-risk HPV DNA typesState the 2 main low-risk HPV DNA types State the 2 main high-risk HPV DNA typesState the 2 main high-risk HPV DNA types State what type of test method is used for the State what type of test method is used for the detection of HPVdetection of HPV State the minimum sample volume required for State the minimum sample volume required for testingtesting State what the molecular sandwich consist ofState what the molecular sandwich consist of Human Papillomavirus (HPV)Human Papillomavirus (HPV) Primary etiological agent in cervical cancerPrimary etiological agent in cervical cancer 2 2nd nd most common type of cancer in women most common type of cancer in women world wideworld wide 3 3rd rd leading cause of cancer-related deaths in leading cause of cancer-related deaths in women worldwidewomen worldwide Most common viral sexually transmitted Most common viral sexually transmitted infection, that goes undiagnosed due to no infection, that goes undiagnosed due to no symptoms developedsymptoms developed HPV CharacteristicsHPV Characteristics Non-enveloped double-stranded DNA virusNon-enveloped double-stranded DNA virus Epitheliotrophic- has great affinity for epithelial Epitheliotrophic- has great affinity for epithelial cellscells Obligatory intracellular parasites that deliver Obligatory intracellular parasites that deliver their genome and accessory proteins into host their genome and accessory proteins into host cells for viral replicationcells for viral replication HPV InfectionHPV Infection E6 oncogene binds to E6 oncogene binds to p53p53 protein in host cell protein in host cell p53p53 protein is a negative regulator protein is a negative regulator E6 protein mutates E6 protein mutates p53p53 protein removing its protein removing its protective functionprotective function Mutation disables Mutation disables p53p53 gene switch, permitting cell gene switch, permitting cell to multiply uncontrolledto multiply uncontrolled HPV TypesHPV Types Types 6 &11most common low-risk HPV Types 6 &11most common low-risk HPV Associated with genital wartsAssociated with genital warts Rarely found in cervical cancerRarely found in cervical cancer Types 16 & 18- most common high-risk HPVTypes 16 & 18- most common high-risk HPV Associated with cancers of the cervix, vagina, vulva, Associated with cancers of the cervix, vagina, vulva, anus, and penisanus, and penis HPV DetectionHPV Detection Traditional screening by Papanicolau Traditional screening by Papanicolau (pap smear) test(pap smear) test Used universally for initial detection of Used universally for initial detection of intraepithelial abnormalitiesintraepithelial abnormalities In case of atypical squamous cells of In case of atypical squamous cells of undetermined significance HPV DNA detection undetermined significance HPV DNA detection is recommendedis recommended .au/./sep/cells_image_inside.jpg Molecular TestingMolecular Testing Hybrid Capture 2 (hc2) System HPV DNA TestHybrid Capture 2 (hc2) System HPV DNA Test Approved by FDAApproved by FDA Nucleic acid hybridization assayNucleic acid hybridization assay No target DNA amplificationNo target DNA amplification Single amplification using microplate Single amplification using microplate chemiluminescence for qualitative detection of 18 chemiluminescence for qualitative detection of 18 types of HPVtypes of HPV /graphics/mags/0409/sl03.jpg Molecular Testing cont.Molecular Testing cont. Differentiates between low and high risk HPVDifferentiates between low and high risk HPV Low-risk HPV: 6, 11, 42, 43, 44Low-risk HPV: 6, 11, 42, 43, 44 High-risk HPV: 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, High-risk HPV: 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59, 6859, 68 Does not determine specific HPV genotypeDoes not determine specific HPV genotype Controls and ReagentsControls and Reagents ControlsControls High-risk control: cloned HPV 16 DNAHigh-risk control: cloned HPV 16 DNA Low-risk control: cloned HPV 6 DNALow-risk control: cloned HPV 6 DNA Negative controlNegative control Carrier DNACarrier DNA Calibrators (run in triplicate)Calibrators (run in triplicate) Low-risk Calibrator: Cloned HPV 11 DNALow-risk Calibrator: Cloned HPV 11 DNA High-risk Calibrator: cloned HPV 16 DNAHigh-risk Calibrator: cloned HPV 16 DNA Ensure that the reagents and calibrator materials are Ensure that the reagents and calibrator materials are functioning properly, for determination of assay cut-off functioning properly, for determination of assay cut-off valuevalue Controls and Reagents cont.Controls and Reagents cont. ProbesProbes Low-risk Probe: HPV 6, 11, 42, 43, 44 RNA cocktailLow-risk Probe: HPV 6, 11, 42, 43, 44 RNA cocktail High-risk Probe: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 58, High-risk Probe: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 58, 59, 68, RNA cocktail59, 68, RNA cocktail Capture MicroplateCapture Microplate Coated with goat polyclonal anti-RNA:DNA hybrid Coated with goat polyclonal anti-RNA:DNA hybrid antibodiesantibodies Detection Reagent 1Detection Reagent 1 Alkaline phosphatase-conjugated murine monoclonal Alkaline phosphatase-conjugated murine monoclonal antibodies to RNA:DNA hybridsantibodies to RNA:DNA hybrids Detection Reagent 2Detection Reagent 2 Chemiluminescent substrateChemiluminescent substrate Specimen RequirementsSpecimen Requirements Cervical specimens collected using a broom type Cervical specimens collected using a broom type collection device placed in PreservCyt Solutioncollection device placed in PreservCyt Solution Cervical biopsies btw 2-5 mm in Digene Cervical biopsies btw 2-5 mm in Digene Specimen Transport MediumSpecimen Transport Medium Specimens collected with the Digene Cervical Specimens collected with the Digene Cervical Sampler, placed in SurePath Preservative FluidSampler, placed in SurePath Preservative Fluid 4mL of sample needed for denaturation process4mL of sample needed for denaturation process Denaturation ProcessDenaturation Process Samples are mixed with Sample Conversion Samples are mixed with Sample Conversion bufferbuffer Then mixed with a 2:1 ratio of Specimen Then mixed with a 2:1 ratio of Specimen Transport Medium (STM) and Denaturation Transport Medium (STM) and Denaturation Reagent (DNR)Reagent (DNR) STM-preservative that retards bacterial growth and STM-preservative that retards bacterial growth and retains DNA integrityretains DNA integrity DNR-dilute sodium hydroxide solution, which lysis DNR-dilute sodium hydroxide solution, which lysis cells and denatures HPV DNAcells and denatures HPV DNA DetectionDetection The Hybrid Capture 2 is a fluid-based molecular The Hybrid Capture 2 is a fluid-based molecular hybridization assayhybridization assay Does not use HPV DNA target amplificationDoes not use HPV DNA target amplification Uses hybridized signal amplificationUses hybridized signal amplification Denatured HPV DNA is hybridized with low-risk or Denatured HPV DNA is hybridized with low-risk or high-risk RNA probehigh-risk RNA probe Resulting hybrids are captured on microplate wells by Resulting hybrids are captured on microplate wells by immobilized antibodyimmobilized antibody DetectionDetection Detection is sandwich-style with a second anti-Detection is sandwich-style with a second anti- RNA:DNA hybrid conjugated to alkaline-RNA:DNA hybrid conjugated to alkaline- phosphatasephosphatase Bound alkaline-phosphatase is revealed by addition Bound alkaline-phosphatase is revealed by addition of a chemiluminescent dioxetane-based substrateof a chemiluminescent dioxetane-based substrate Substrate is cleaved by bound alkaline phosphate Substrate is cleaved by bound alkaline phosphate and emitted light is measured in a microplate and emitted light is measured in a microplate luminometerluminometer www.papillomavirus.cz/images/hybridcapture.jpg Test InterpretationTest Interpretation Emitted light is measured in Relative Light Units Emitted light is measured in Relative Light Units (RLUs)(RLUs) Specimens with RLU/CO ratio 1.7 with low-risk Specimens with RLU/CO ratio 1.7 with low-risk HPV probe are considered positive for low-risk HPVHPV probe are considered positive for low-risk HPV Specimens with RLU/CO ratio 1.7 with high-risk Specimens with RLU/CO ratio 1.7 with high-risk HPV probe are considered positive for high-risk HPVHPV probe are considered positive for high-risk HPV Specimens with RLU/CO ratio btw 0.80-1.7 are Specimens with RLU/CO ratio btw 0.80-1.7 are considered indeterminate for either low-risk or high-considered indeterminate for either low-risk or high- risk HPV and must be repeatedrisk HPV and must be repeated LimitationsLimitations Significant number of false-positives (10%-19%) Significant number of false-positives (10%-19%) due to cross reactivity with low-risk HPV DNAdue to cross reactivity with low-risk HPV DNA HPV DNA not amplifiedHPV DNA not amplified Negative predicted value may be compromised in Negative predicted value may be compromised in cases in which HPV DNA copy number is lowcases in which HPV DNA copy number is low No internal control used for sample sufficiencyNo internal control used for sample sufficiency Not possible to determine if results are due to Not possible to determine if results are due to insufficient DNA or true negativeinsufficient DNA or true negative Large number of inconclusive resultsLarge number of inconclusive results Requires large sample volumeRequires large sample volume Sources of ErrorSources of Error Large concentrations of whole blood, douche, anti-Large concentrations of whole blood, douche, anti- fungal cream, and contraceptive jellyfungal cream, and contraceptive jelly May cause false-negativeMay ca