Development of a real-time quantitative RT-PCR to detect REV contamination in live vaccine

• Published in: Poultry science 2016-09, Vol.95 (9), p.2023-2029
• Main Authors: Luan, Huaibiao, Wang, Yixin, Li, Yang, Cui, Zhizhong, Chang, Shuang, Zhao, Peng
• Format: Article
• Language: English
• Subjects: Animals; Gene Products, pol - isolation & purification; Poultry Diseases - prevention & control; Poultry Diseases - virology; Real-Time Polymerase Chain Reaction - veterinary; Reticuloendotheliosis virus - immunology; Reticuloendotheliosis virus - isolation & purification; Retroviridae Infections - prevention & control; Retroviridae Infections - veterinary; Retroviridae Infections - virology; Tumor Virus Infections - prevention & control; Tumor Virus Infections - veterinary; Tumor Virus Infections - virology; Vaccines, Attenuated - analysis; Viral Vaccines - analysis
• ISSN: 0032-5791; 1525-3171
• DOI: 10.3382/ps/pew147

Based on the published Avian reticuloendotheliosis virus (REV) whole genome sequence, primers and TaqMan probes were designed and synthesized, and the TaqMan probe fluorescence real-time quantitative RT-PCR (qRT-PCR) method for detecting the REV pol gene was established by optimizing the reaction conditions. Sensitivity analysis showed that the qRT-PCR method had a sensitivity that was 1,000-fold higher than conventional PCR. Additionally, no amplification signals were obtained when we attempted to detect DNA or cDNA of ALV-A/B/J, MDV, CIAV, IBDV, ARV, NDV, AIV, or other viruses, suggesting a high specificity for our method. Various titers of REV were artificially “spiked” into the FPV and MDV vaccines to simulate REV contamination in attenuated vaccines to validate this qRT-PCR method. Our findings indicated that this qRT-PCR method could detect REV contamination at a dose of 1 TCID50/1,000 feathers, which was 10,000-fold more sensitive than the regular RT-PCR detection (104 TCID50/1000 feathers).