USEFULNESS OF AN MULTIPLEX REALTIME PCR FOR DETECTION OF DIFFERENT CAPRINE ARTHITIS VIRUS GENOTYPES

Introduction: A serological survey carried out between 2005 and 2008 by the “Istituto Zooprofilattico Sperimentale” of Sardinia; showed that CAEV infection is spread throughout the regional territory with a prevalence of positive farms around 80% and a seropositivity of more than 70% of tested animals. The CAE is a serious threat to goat-sheep heritage and is likely to compromise an economy that is a fundamental source of income for breeders, especially in those areas considered "agronomically" difficult. It can be inferred therefore that the disease will become more important in the coming years than it has been in the past. Currently, there are no vaccines for the control of the infection, thus the only prophylaxis that can be implemented is hygienic- sanitary. Prevention measures are based on the removal of healthy infected heads. In this context, laboratory testing for CAEV-positive samples plays a crucial role in the entire diagnostic procedure. The aim of the work was to evaluate a molecular system based on real-time PCR amplification on relatively gag gene sequences in the SRLV, as already indicated by several authors [1,2]. Methods: A total of 50 blood samples from farms in southern Sardinia with a clinical diagnosis for CAEV infections have been anlyzed and proviral DNA was obtained from buffy-coat by GeneProof Pathogen Kit (Brno-Czech Republic), 2 μl of DNA extract was used in a real-time PCR reaction by using the kit SYBR Premix (TaKara-Clontech ®) with a LightCycler II Roche® apparatus following the manufacture istructions [3]. The PCR target region was identified by multiple sequence alignment program Geneious Biomatters Ltd., on gag gene sequences available in the DNA data Bank (GenBank). Melting curve analysis showed two different peaks after real time PCR reaction. Positive samples showed a Tm peak ranged from 82 °C to 88 °C, while the negative sample was identified for a unique peak positioned from 73 °C to 77 °C, Figure 1. These samples resulted 90% CAEV positives and the analysis of the melting curve revealed several multiple-infection (different CAEV variants in the same subject), in this case the most detected sequences are referenced to genotypes B3 and E (subtype Volterra, Fonni, Roccaverano, Seui) GenBank accession n. JF502417, JF502416, EU293537, GQ381130 Conclusions: The presented method/approach could be suitable for a rapid and complete laboratory diagnosis of CAEV infection.