! "!
ABSTRACT.
!
Norovirus (NoV), a member of Caliciviridae family, are causative agents of acute non bacterial gastroenteritis and are widespread into the environment at high concentrations. They can be divided into five genetically distinct genogroups:
genogroup I (NoV GI) and genogroup II (NoV GII) are responsible for the majority of human clinical cases. The hepatitis A virus (HAV) belongs to the genus Hepatovirus, family Picornaviridae, and it is recognized as an important cause of acute viral hepatitis in humans worldwide. NoV and HAV, stable and resistant to inactivation in the environment, are transmitted particularly by fecal-oral route, cause frequently health risks due to contamination of foods, such as bivalve shellfish, especially if eaten raw or undercooked.
The molecular diagnosis of these viruses in bivalve shellfish showed several problems in these matrices both because of low levels of contamination and the difficulty of obtaining efficient extraction. The presence of PCR inhibitors may also affects the efficiency of amplification of the Real Time RT-PCR.
In this paper a comparison was made between two different methods of viral RNA extraction from bivalve shellfish in order to evaluate their extraction efficiencies, using a co-extracted process control (FCV), and co-amplified amplification control (NoV GII viral RNA).
Twenty-four samples of hepatopancreas of Mytilus edulis, Donax trunculus, Crassostrea gigas and twelve samples of Chamelea gallina were contaminated with FCV and the viral RNA was extracted with Kit 1 (QIAamp Viral RNA Mini Kit, QIAGEN) and Kit 2 (NucliSENS® Minimag®, bioMérieux). The RNA, extracted with
! "!
two different methods, were examined by Real time RT-PCR to evaluate the efficiency of extraction and amplification.
In order to construct positive controls and to develop a quantitative Real Time RT- PCR, plasmids containing NoV GI, NoV GII and HAV were prepared from reverse- transcribed PCR products
The comparison between the two kits on different matrices showed significant differences, and, in particular, Kit 1 resulted more efficient than Kit 2 in this kind of samples. Moreover, high variability of both efficiencies was showed between the different matrices extracted by the use of the two kits.
Transcripts produced from plasmids containing the NoV GI, NoV GII and HAV fragment insertions allowed to develop a quantitative method.
