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Pesquisa Veterinária Brasileira
On-line version ISSN 1678-5150
SIMIONATTO, Simone; LIMA-ROSA, Carlos André da Veiga; RUBIN, Lauricio Librelotto and CANAL, Cláudio Wageck. A nested-PCR protocol for detection of the chicken anemia virus. Pesq. Vet. Bras. [online]. 2005, vol.25, n.2, pp.106-110. ISSN 1678-5150. http://dx.doi.org/10.1590/S0100-736X2005000200007.
This paper reports a nested polymerase chain reaction (nested-PCR) protocol for detection of chicken anemia virus (CAV), the causal agent of infectious chicken anemia. For DNA extraction from clinical samples, a method based on guanidine thiocyanate was found more sensitive and practical than other extraction protocols tested. The pair of primers used in the initial PCR targeted a 664 bp fragment on the VP1 gene. The primers for the internal PCR targeted a fragment of 520 bp. The specificity of the primers was evaluated on samples of CAV controlled flocks. Thirty different viruses and bacteria isolated from chickens did not give rise to any amplification product in the assay. The sensitivity of the nested-PCR was determined on serial dilutions of a CAV vaccine. The nested-PCR was more sensitive than a one step PCR and was able to detect at least 0.16 TCID50 of the vaccine strain. In addition, the protocol employed here detected viral DNA from tissues, sera and litter from flocks with or without clinical signs of disease. It is concluded that the nested-PCR protocol described here is more sensitive, faster and less cumbersome than virus isolation in cell culture as a diagnostic technique for detection of CAV.
Keywords : Chicken anemia virus; CAV; molecular diagnosis; nested-PCR.