The subject of this dissertation is the work I carried out at AB ANALITICA s.r.l. in the context of a doctoral program based on higher apprenticeship, an innovative form of PhD aiming at reducing the distance between academia and business.
In the first part of my PhD I worked on the new version of a product for HPV detection, recently developed at AB ANALITICA: the REALQUALITY RI-HPV STAR device. My task was to bring the original device to a higher level of analytical sensitivity for certain HPV genotypes as required by the World Health Organization (WHO).
This device was based on Real-time PCR and melting curve analysis with intercalating dye technology. The primers used in the original reaction mix were GP5+/GP6+ primers. These primers are capable of detecting most HPV types in aggregate. However, the analytical sensitivity they provide is not equal for all the detected genotypes and their performance was not in compliance with WHO requirements for genotyping tests. Thus, first of all, the primer composition in the reaction mix was modified by adding other primers to the original pair. A multiple primer reaction required employing specific reagents and, at the end, the whole set up of the assay had to be modified. The occasion was used to also improve an ergonomic aspect and amplify the target pathogen and the internal control gene in multiplex instead of running two separate reactions as in the original assay.
Higher analytical sensitivity was achieved, but primer dimers related issues emerged, which could have impaired the correct interpretation of the assay results, and the project was abandoned.
In the second part of my PhD, I worked at the design, development and validation of two new assays for HPV detection, based on Real-time PCR with target specific primers and TaqMan fluorescent probes.
The HPV types detected by the first of the two assays are the 14 most common high risk types, i.e. HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. The second assay detects the two most common low risk types, i.e. HPV 6, 11, as well as other possible high risk types, i.e. HPV 26, 53, 67, 70, 73 and 82.
Assay 1 has already been validated and its performance resulted largely in compliance with WHO requirements and with the acceptability criteria established in the validation plan. This assay, therefore, is ready to be commercialized under the name REALQUALITY RQ-HPV HR MULTIPLEX.
As for assay 2, some performance parameters have still to be assessed; however, initial data are promising. This second assay, if associated to assay 1, provides the final user with a complete panel for HPV investigation. The two assays combined, therefore, will be soon proposed, as an alternative product, under the name REALQUALITY RQ-HPV HR/LR MULTIPLEX
