Development of TaqMan Real-Time Polymerase Chain Reaction Assay for Detection of Chicken Anemia Virus in Newcastle Disease Vaccines.

Abstract

Exogenous chicken anemia virus (CAV) has been detected in commercial poultry vaccines in various countries of the world. The presence of unwanted CAV in vaccines not only influences the epidemiology of chicken infectious anemia disease, but may also lead to vaccine failure and confusing results when vaccine responses are monitored. To detect CAV in contaminated vaccines, nucleic acid testing (unlike conventional testing) has a shorter processing time and does not require cell culture or live animals. The aim of the current study was to develop a TaqMan real-time polymerase chain reaction (PCR) assay to detect and quantify CAV in poultry vaccines and investigate CAV contamination in Razi live Newcastle disease vaccines. The TaqMan real-time PCR assay was set up, optimized, and validated in successive experiments. A standard plasmid pUC-VP2 containing viral protein 2 of CAV was constructed and used in the assay to generate a standard curve to quantify CAV genomes. A clear linear correlation was observed between threshold cycle (Ct) values and plasmid copy numbers in the amplification plots of 10-fold serial dilution of the plasmid. Total DNA of three samples of each of four different Razi live Newcastle disease vaccines, namely LaSota, B1, clone.12IR, and thermo-resistant strains, were extracted and subjected to real-time PCR assay. No CAV contamination was detected in the Razi Live Newcastle vaccines. The developed TaqMan real-time PCR assay provides a quick, specific, and sensitive method for use in detecting CAV in quality control vaccine testing and viral load studies.