Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.
Arch Razi Inst. 2021 Sep 1;76(3):421-427. doi: 10.22092/ari.2020.342677.1474. eCollection 2021 Summer.
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.
外源性鸡贫血病毒（CAV）已在世界各国的商业家禽疫苗中被检测到。疫苗中存在不需要的 CAV 不仅会影响鸡传染性贫血病的流行病学，还可能导致疫苗失效，并在监测疫苗反应时导致结果混乱。为了在污染的疫苗中检测 CAV，核酸检测（与常规检测不同）具有更短的处理时间，并且不需要细胞培养或活体动物。本研究的目的是开发一种 TaqMan 实时聚合酶链反应（PCR）检测方法，用于检测和定量家禽疫苗中的 CAV，并调查 Razi 活新城疫疫苗中的 CAV 污染情况。在连续的实验中建立、优化和验证了 TaqMan 实时 PCR 检测方法。构建了含有 CAV 病毒蛋白 2 的 pUC-VP2 标准质粒，并在检测中用于生成标准曲线以定量 CAV 基因组。在质粒 10 倍系列稀释的扩增图谱中，阈值循环（Ct）值与质粒拷贝数之间观察到清晰的线性相关性。从四种不同的 Razi 活新城疫疫苗（即 LaSota、B1、clone.12IR 和耐热株）的每个样本中提取总 DNA，并进行实时 PCR 检测。在 Razi 活新城疫疫苗中未检测到 CAV 污染。开发的 TaqMan 实时 PCR 检测方法为在质量控制疫苗检测和病毒载量研究中检测 CAV 提供了一种快速、特异和敏感的方法。
