Development of a real-time quantitative RT-PCR to detect REV contamination in live vaccine.
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 (10(4) TCID50/1000 feathers).
College of Veterinary Medicine, Shandong Agricultural University, Tai'an, 271018.
Huaibiao
H
Luan
College of Veterinary Medicine, Shandong Agricultural University, Tai'an, 271018.
Yixin
Y
Wang
College of Veterinary Medicine, Shandong Agricultural University, Tai'an, 271018.
Yang
Y
Li
College of Veterinary Medicine, Shandong Agricultural University, Tai'an, 271018.
Zhizhong
Z
Cui
College of Veterinary Medicine, Shandong Agricultural University, Tai'an, 271018 changshuang81@sdau.edu.cn zhaopeng@sdau.edu.cn.
Shuang
S
Chang
College of Veterinary Medicine, Shandong Agricultural University, Tai'an, 271018 changshuang81@sdau.edu.cn zhaopeng@sdau.edu.cn.
Peng
P
Zhao
D000818:Animals; D015684:Gene Products, pol / Q000302:isolation & purification*; D011201:Poultry Diseases / Q000517:prevention & control* / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary*; D001356:Reticuloendotheliosis virus / Q000276:immunology* / Q000302:isolation & purification; D012192:Retroviridae Infections / Q000517:prevention & control / Q000662:veterinary* / Q000821:virology; D014412:Tumor Virus Infections / Q000517:prevention & control / Q000662:veterinary* / Q000821:virology; D014613:Vaccines, Attenuated / Q000032:analysis; D014765:Viral Vaccines / Q000032:analysis*
D015684:Gene Products, pol; D014613:Vaccines, Attenuated; D014765:Viral Vaccines
contamination; detection; live vaccine; real-time RT-PCR; reticuloendotheliosis virus (REV)