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Development of a semi-nested PCR using degenerate primers for the generic detection of small ruminant lentivirus proviral DNA

2006; Elsevier BV; Volume: 135; Issue: 2 Linguagem: Inglês

10.1016/j.jviromet.2006.03.010

1879-0984

Yassir Mohammed Eltahir, Chrysostomos Ι. Dovas, Maria Papanastassopoulou, M Koumbati, Nektarios D. Giadinis, S. Verghese-Nikolakaki, G. Koptopoulos,

Herpesvirus Infections and Treatments

A PCR assay was developed for the reliable detection of small ruminant lentivirus (SRLV) proviral DNA. The method involved the use of degenerate deoxyinosine-substituted primers and a second semi-nested PCR step that increased the polyvalency and sensitivity of the detection, respectively. Primers were designed from the pol gene conserved motifs of 85 SRLV isolates and were evaluated using different SRLV isolates together with Maedi-Visna virus (MVV) and caprine arthritis–encephalitis virus (CAEV) reference strains. The method successfully detected SRLV proviral DNA in total DNA extracts originating from whole blood samples, separated peripheral blood mononuclear cells (PBMCs) and tissue cultures. The semi-nested PCR was compared with the agar gel immunodiffusion test and proved to be highly sensitive, specific and capable of detecting many SRLV variants in infected or suspect animals. Therefore, it would be useful in the diagnosis of natural SRLV infections, in eradication programs and epidemiological studies. Whole blood samples can be used directly, thus alleviating the need for PBMC separation, and thereby enables a simple, fast and cost-effective analysis of a large number of samples.