Reaction in Chain of Polymerase (PCR) for detention of the genes gag and pol of the Immunodeficiency Virus, Feline (FIV)
The goal of the present work was to standardize the polymerase chain reaction (PCR) technique to detect feline immunodeficiency virus (FIV) proviral genome.FIV is a retrovirus associated with AIDS-like illnesses in cats. We studied 97 domestic cats blood samples from Rio de Janeiro city. From these, 60 were seropositive for FIV and 37 were seronegative, included as negative control group.Their immunological status was evaluated by enzyme immune assay (EIA ? FIV / FeLV SNAPamp;#61650; COMBO). Among the seropositive cats, 40 (67%) presented clinical symptoms. Thirty eight cats were classified in complex related to AIDS (CRA), and 2 were classified into AIDS phase. Viral DNA was extracted from blood samples using QIAmp Blood Mini Kit (QIAGEN). Oligonucleotide primers for a mithochondrial DNA copy of the 16s gene were utilised as positive control to test DNA integrity.To detect FIV proviral genome the pair of primers targeted specifically at regions of 483 bp gag gene and 471 bp pol gene.The primers for the internal PCR targeted at fragments of 203 bp gag gene and 183 bp pol gene.The specificity of the primers was evaluated on samples from feline leukemia virus (FeLV) infected cats. FeLV is a heterologous retrovirus. To reach the best amplification method, four different amplification protocols were evaluated for the gag region and three for the pol region. In the seropositive cats group, 39 (65%) presented proviral DNA amplification to gag region using four protocols, and 6 (10%) presented proviral DNA amplification to pol region using three protocols. Among the seronegative cats, proviral DNA gag region was detected in one sample.In this case, serological conversion was seen in another sample collected subsequently. The PCR fragments were identified through the sequencing analysis in 8 samples.However, inconclusive results were obtained related to viral subtypes. protocols selected for each region showed statistical significant results only for gag region. The nested PCR proved to be an useful tool for the FIV study, presenting an efficient amplification proviral DNA from FIV seropositive cats blood samples. This methodology is related to specific situations such as no detectable antibodies induced by viral infection and it can be used as an alternative technique to confirm viral diagnosis and early FIV infection identification.
Advisor:Hermann Gonçalves Schatzmayr
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Reaction in chain of Polymerase
Date of Publication:08/02/2006