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Title:DEVELOPMENT OF A NOVEL PCR-BASED ASSAY FOR HIGH-RISK HUMAN PAPILLOMAVIRUS DETECTION AND GENOTYPING IN SELF COLLECTED CERVICOVAGINAL SAMPLES: A NEW POSSIBILITY FOR THE CERVICAL CANCER SCREENING
Authors:Avian, Alice (Author)
Marini, Bruna (Mentor) More about this mentor... New window
Files:.pdf Alice_Avian.pdf (37,88 MB)
 
Language:English
Work type:Doctoral dissertation (mb31)
Tipology:2.08 - Doctoral Dissertation
Organization:FPŠ - Graduate School
Abstract:Human Papillomavirus (HPV) infection is the causative agent for the invasive cervical cancer and its precancerous lesions, furthermore, there are growing evidences of HPV being a relevant factor in other anogenital cancers as well as head and neck cancers. Most sexually active women become infected with HPV at least once in their lifetime, but less than 10% of women becomes persistently infected, and it is precisely the persistent infection that contributes to the development of cervical cancer. The preventive effect of cervical cancer screening largely depends in the high women participation and coverage; indeed, a large number of cervical cancers diagnoses normally arise among under-screened and unscreened women. Increase in the screening coverage is essential to improve the effectiveness of cervical screening programmes. The main purpose of this PhD project was to solve some of the most relevant problems in the cervical cancer screening programmes, as the increase of cost-effectiveness and the amelioration of the screening coverage. My work was focused on the development and validation of the first Ulisse BioMed S.p.A. product, the HPV Selfy™ test, an innovative PCR-based kit for the direct detection and genotyping of 12 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) and 2 possible/probable high-risk (66 and 68), specifically optimized for the analysis of self-collected vaginal specimens. The core of this innovative test is based on high-resolution melting (HRM) analysis, a recently developed technique for fast, high-throughput post-PCR analysis of variance in nucleic acid sequences, that characterizes the amplicons by studying thermal denaturation of double-stranded DNA. Based on this approach and through the design of different HPV type-specific primer pairs and the development of a specific master mix, unique melting peaks in a single fluorescence channel were obtained, allowing the multiple detection and genotyping of 14 HPV types in a single PCR well. Three different clinical studies have been carried out to validate the assay on the vaginal self-collected samples with truly amazing results regarding the assay’s performance, but also for self-sampling acceptability by women. Moreover, data collected in these studies suggest a future possible use of this test for the hard-to-reach women, as an alternative of the conventional clinician-collected sample, in order to increase the cervical cancer screening coverage.
Keywords:Human Papillomavirus, HPV test, cervical cancer screening, prevention, diagnostic test, High resolution melting, HRM, genotyping, PCR, Self-sampling, clinical validation.
Year of publishing:2020
Source:Nova Gorica
COBISS_ID:19975939 Link is opened in a new window
URN:URN:SI:UNG:REP:XNZPU4WP
Views:371
Downloads:7
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Secondary language

Language:Slovenian
Title:RAZVOJ NOVEGA TESTA NA OSNOVI PCR ZA ODKRIVANJE IN GENOTIPIZACIJO VISOKORIZIČNIH TIPOV HUMANIH VIRUSOV PAPILOMA V CERVIKOVAGINALNIH VZORCIH: NOV PRESEJALNI PRISTOP PRI RAKU MATERNIČNEGA VRATU
Abstract:Humani virusi papiloma (HPV) so povzročitelji invazivnega raka materničnega vratu in njegovih predrakavih lezij, pomembno pa prispevajo tudi k zavoju drugih anogenitalnih rakov ter raka glave in vratu. Večina spolno aktivnih žensk se vsaj enkrat v življenju okuži z virusom HPV, pri manj kot 10% žensk pa se pojavi dolgotrajna okužba s HPV, ki je ključna za razvoj raka materničnega vratu. Preventivni učinek presejanja raka materničnega vratu je v veliki meri odvisen od sistematične udeležbe in visoke stopnje pokritosti presejalnih testov. Največje število novo odkritih primerov raka materničnega vratu je prav pri ženskah, ki so presejalne teste opravljale redko ali nikoli. Izboljšanje pokritosti s presejalnimi testi je zato ključnega pomena za večjo učinkovitost presejalnih programov za ugotavljanje raka materničnega vratu. Glavni namen doktorskega projekta je bil prispevati z reševanju dveh najpomembnejših težav v presejalnih programih raka materničnega vratu, in sicer povečanju stroškovne učinkovitosti in izboljšanju presejalne pokritosti. Doktorsko delo je bilo osredotočeno na razvoj in validacijo prvega izdelka Ulisse BioMed SpA, testa HPV Selfy™, inovativnega kompleta na osnovi PCR za neposredno odkrivanje in genotipizacijo 12 visokorizičnih tipov HPV (16, 18, 31, 33 , 35, 39, 45, 51, 52, 56, 58 in 59) in 2 verjetno visokorizična tipa (66 in 68), posebej optimiziranega za analizo samovzorčenih vaginalnih vzorcev. Jedro tega inovativnega testa je analiza na podlagi talitvene krivulje (HRM). Ta nedavno razvita metoda za hitro, visoko-pretočno analizo variabilnosti v zaporedjih nukleinskih kislin, razvrsti amplikone na osnovi termične denaturacije dvojno-verižne DNA. Razvili smo pare oligonukleotidnih začetnikov, specifičnih za različne tipe HPV, in posebno reakcijsko mešanico. Ta metoda omogoča ločevanje talitvenih vrhov v enem fluorescenčnem detekcijskem kanalu, zaradi česar lahko odkrijemo in genotipiziramo 14 različnih tipov HPV v eni sami reakciji PCR. Za validacijo testa na vaginalno odvzetih vzorcih so bile opravljene tri različne klinične študije z resnično dobrimi rezultati glede uspešnosti testa, pa tudi visoke sprejemljivost samo-vzorčenja pri ženskah. Podatki kliničnih študij potrjujejo primernost tega testa za vzorčenje vaginalnih vzorcev pri ženskah, ki se na testiranje z odvzemom kliničnih vzorcev pri zdravniku slabo odzivajo. Test bi lahko predstavljal dobro alternativo standardnemu testiranju z namenom izboljšanja pokritosti presejalnega testiranja raka materničnega vratu.
Keywords:humani virusi papiloma, test HPV, testiranje raka materničnega vratu, preventiva, diagnostični test, HRM, genotipizacija, PCR, samovzorčenje, klinično testiranje.


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