Sviluppo di una tecnologia Q-LAMP quantitativa mediante la messa a punto di un nuovo modello di saggio per la rilevazione e la quantificazione del virus Epstein-Barr.

Epstein-Barr virus (EBV) also called human herpesvirus 4 (HHV-4) is a double-stranded DNA viruses that infects B cells. The genome is about 172 kb in length and encodes for more than 80 genes. EBV is classified into two subtypes: Type 1 / A and 2 / B depending on the sequence of the EBNA-2 gene. More than 90% of the world’s population has been exposed to the EBV infection, like all herpes viruses it is able to persist in the host for life with a silent infection by integrating in its genome. The quantification of EBV DNA loads in blood, plasma, serum and CSF (cerebrospinal fluid) is essential for the diagnosis of chronic infection of EBV, especially for the diagnosis of EBV-associated lymphoproliferative disorders in immunocompromised subjects. Currently serological assay and methods PCR-based has been used for the detection of EBV. Both of these are time consuming and need more steps for the set-up, therefore require specialized personnel and equipped laboratories. This thesis describes the development of a quantitative Q-LAMP technology generating a new model assay for the detection and quantification of the two most clinically relevant subtypes of EBV virus. Initially the Q-LAMP technology has been developed only for qualitative purposes thanks to its features to be simple, rapid, specific and sensitive, therefore it is easily applicable in the clinical laboratories by using basic facilities. The Q-LAMP EBV assay has been designed using the qualitative Q-LAMP properties and has been improved to be quantitative, high sensitive, specific and accurate on four different matrices (whole blood, plasma, serum and CSF). The optimization has been carried out both on plasmids and on negative extracted matrices and the final assay has been validated on clinical samples. Thanks to the multiplex format it has been possible to introduce in the reaction a control that allows the assessment of the sample extraction and the validation of the negative result. The amplification and the detection of the target and the internal control occur in real-time in a single tube. To evaluate the diagnostic performance and the feasibility in clinical practice, the assay has been validated on positive and negative clinical samples compared with the standard method Real-Time PCR using as extraction method the Liaison IXT platform (DiaSorin). The correlation between the two methods on whole blood and plasma is acceptable and meet the requests of corporate guidelines, made in response to the market trend. These results have been demonstrated that Q-LAMP EBV assay is suitable for industrialization for a future diagnostic product.