Validation of polymerase chain reaction assay as an alternative method for detection of chicken anemia virus as a vaccine contaminant.

Freedom of veterinary vaccines from extraneous virus contamination is one of the most important requirements for vaccine before release. Testing of vaccines for detection of extraneous virus contamination is currently achieved in the Central Laboratory for Evaluation of Veterinary Biologics (CLEVB) by conventional in - vitro and / or in - vivo assays as tissue culture inoculation and chicken inoculation, which are time consuming and laborious. In this study, the polymerase chain reaction (PCR) was used as an alternative technique – for detection of chicken anemia virus (CAV) contamination in avian live viral vaccines. Specificity of the assay was verified by detection of CAV specific product only in templates extracted from CAV preparations, but not detected in templates of various avian viruses including Newcastle dis-ease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), avian Reovirus (ARV), fowl pox virus (FPV), avian encephalomyelitis virus (AEV) and Marek´s disease virus (MDV). Assay sensitivity was evaluated by testing serial 10- fold dilutions of CAV stock, and the determined detection limit was high enough to detect 40 TCID50 of CAV per reaction. Another set of CAV dilutions were prepared in three different types of avian live vaccines, namely NDV, ARV , and FPV vaccines( the vaccine matrix was used as diluents), and PCR was performed to study the effect of vaccine matrix on reaction sensitivity as well as to verify the assay efficacy in detection and recovery of CAV in contaminated vaccines. Results indicated that PCR sensitivity was not affected by the presence of NDV, or ARV matrices. However, the assay sensitivity was decreased by one log in case of Fowlpox vaccine matrix. This PCR assay could be used as an alternative technique to the time- consuming, tedious, in vivo and in vitro assays, and application of this technique can facilitate the routine testing of vaccines for detection of CAV as an extraneous virus contaminant.

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