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Militello, Valentina (2013) Sviluppo di un metodo accurato per l'identificazione e genotipizzazione del papillomavirus umano basato sul sequenziamento 454 di nuova generazione. [Tesi di dottorato]

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Abstract (inglese)

Accurate HPV typing is essential for evaluation an monitoring HPV vaccines, as second line testing in cervical cancer screening and in epidemiological surveys. In this study, we set up and assessed in clinical samples a new HPV typing method based on 454 next-generation sequencing (NGS) of HPV L1 amplicons. We set up a first protocol by using modified degenerated primer MY09/11 to analyze 164 cervical samples. This protocol represent a proof of principle that NGS can be used for HPV typing, but had an intrinsic weakness conceivably due to mismatches between the sequence of the MY09/11 primers. For this reason we generate a new protocol by using a modified PGMY primer set with improved sensitivity for some HPV types that are not targeted by standard PGMY primers. By using a median 12,000-fold coverage, the NGS method allowed to correctly identify all high-risk HPV types, either in single and multiple infection, with a sensitivity of 50 genome equivalents, as demonstrated by testing WHO LabNet EQA sample panels. Analysis of mixtures of HPV16- and HPV18-positive cell lines demonstrated that the NGS method could reproducibly quantify the proportion of each HPV type in multiple infections in a wide dynamic range. Testing of HPV-positive clinical samples showed that NGS could correctly identify a high number of HPV types in multiple infections. The NGS method was also effective in the analysis of a set of cervical specimens with discordant results at hybrid capture 2 and line probe assays. In conclusion, a new HPV typing method based on 454 pyrosequencing was set up. This method was sensitive, specific, quantitative, and precise in both single and multiple infections. It could identify a wide range of HPV types and might potentially discover new HPV types and variants.

Abstract (italiano)

Un' accurata tipizzazione di HPV è essenziale per la valutazione e il monitoraggio della vaccinazione, come test di secondo livello nello screening del carcinoma cervicale e per le indagini epidemiologiche. In questo studio, è stato messo a punto e testato su campioni clinici un nuovo metodo di tipizzazione di HPV basato sul sequenziamento 454 next-generation (NGS) dell' amplicone L1 di HPV.
A tale scopo è stato sviluppato un primo protocollo che utilizzava dei primer ottenuti modificando i primer degenerati MY09/11; tale protocollo è stato testato su 164 campioni citologici cervicali fornendo una prima dimostrazione che il NGS può essere utilizzato per la tipizzazione di HPV. Il protocollo presentava però alcuni limiti legati soprattutto al disegno dei primer. E' stato quindi studiato un secondo protocollo. Tale protocollo utilizzava un set di primer PGMY modificati in modo da ottenere sensibilità migliorata per alcuni tipi di HPV che non erano riconosciuti dai primer PGMY standard.
Usando una median coverage di 12,000-fold, il metodo NGS è arrivato a identificare correttamente tutti i tipi di HPV ad alto rischio sia in infezioni singole che multiple, con una sensibilità di 50 genomi equivalenti, come dimostrato testando il pannello di campioni del WHO LabNet EQA. L' analisi di miscele di linee cellulari positive per HPV16 e per HPV18 ha dimostrato che il metodo NGS può quantificare in modo riproducibile la proporzione con cui ogni tipo virale è presente nelle infezioni multiple con un ampio range. Nell'analisi di campioni clinici il metodo NGS ha identificato correttamente un ampio numero di tipi di HPV nelle infezioni multiple ed è stato anche utile nell'analisi di campioni cervicali con risultati discordanti al test hybrid capture 2 e line probe assays. Il metodo ha permesso inoltre di riconoscere la presenza di varianti intratipo (sostituzioni nucleotidiche) nello stesso campione clinico. In conclusione, è stato messo a punto un nuovo metodo di tipizzazione dell' HPV basato sul pirosequenziamento 454. Questo metodo è sensibile, specifico, quantitativo, e preciso sia nelle infezioni singole che nelle infezioni multiple. Permette inoltre di identificare un ampio range di tipi di HPV e potenzialmente consente di scoprire nuovi tipi di HPV e varianti.

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Tipo di EPrint:Tesi di dottorato
Relatore:Barzon, Luisa
Correlatore:Barzon, Luisa
Dottorato (corsi e scuole):Ciclo 25 > Scuole 25 > BIOMEDICINA
Data di deposito della tesi:28 Gennaio 2013
Anno di Pubblicazione:28 Gennaio 2013
Parole chiave (italiano / inglese):papillomavirus, genotyping, ultra deep sequencing
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/07 Microbiologia e microbiologia clinica
Struttura di riferimento:Dipartimenti > Dipartimento di Medicina Molecolare
Codice ID:5555
Depositato il:19 Nov 2013 10:33
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