Establishment of a TaqMan-based real-time quantitative PCR method for detection of exogenous fowl adenovirus type Ⅰ, type Ⅲ and avian leukosis virus in human cold adapted live attenuated influenza vaccines
Min Wu,
Baihui Zhi,
Rui Sheng,
Entong Zhou,
Qian Zhou,
Jiemin Li,
Xiaoshu Fu,
Yanchun Wu,
Yunchang Zhang,
Pingjie Sun,
Wei Jia,
Liang Shi,
Dawei Liu,
Hui Zhao,
Weiheng Su
Affiliations
Min Wu
National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, 130012, China
Baihui Zhi
BCHT Biotechnology Company, Changchun, 130012, China
Rui Sheng
National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, 130012, China
Entong Zhou
National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, 130012, China
Qian Zhou
Beijing Vital River Laboratory Animal Technology Company, Beijing, 100012, China
Jiemin Li
National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, 130012, China
Xiaoshu Fu
National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, 130012, China
Yanchun Wu
BCHT Biotechnology Company, Changchun, 130012, China
Yunchang Zhang
BCHT Biotechnology Company, Changchun, 130012, China
Pingjie Sun
BCHT Biotechnology Company, Changchun, 130012, China
Wei Jia
BCHT Biotechnology Company, Changchun, 130012, China
Liang Shi
BCHT Biotechnology Company, Changchun, 130012, China
Dawei Liu
BCHT Biotechnology Company, Changchun, 130012, China
Hui Zhao
National Institutes for Food and Drug Control, Beijing, 102629, China; Corresponding author. National Institutes for Food and Drug Control, Beijing 102629, China.
Weiheng Su
National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, 130012, China; Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, School of Life Sciences, Jilin University, Changchun, 130012, China; Corresponding author. National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, 130012, China.
Cold adapted live attenuated influenza vaccines can effectively prevent human disease and death caused by influenza virus. Since chicken embryos are used as the culture substrate for the large-scale production of influenza vaccines, cold adapted live attenuated influenza vaccines may be contaminated by exogenous avian viruses. Rapid and sensitive methods such as TaqMan-based quantitative PCR are needed for the detection of exogenous avian viruses during cold adapted live attenuated influenza vaccines production. In this study, a TaqMan-based quantitative PCR method was established for the detection of three common exogenous avian viruses, including fowl adenovirus type I, type Ⅲ and avian leukosis virus. Avian virus-encoding plasmids purified in high-performance liquid chromatography were essential for sensitivity analysis. The sensitivity reached 1 copy per reaction for each of the avian virus plasmids. Standard curves showed a strong linear relationship. The TaqMan-based quantitative PCR method had high specificity and no cross-reactivity with other irrelevant viruses. Furthermore, the established TaqMan-based quantitative PCR can effectively detect 0.1 TCID50 of each avian virus without or with interference from the influenza virus nucleic acid. Ultimately, this method was used to test three master seed lots of monovalent cold adapted live attenuated influenza vaccine, and the results showed that no fowl adenovirus type I, type Ⅲ or avian leukosis virus contamination, which were consistent with serological methods. The TaqMan-based quantitative PCR method for the determination of extraneous avian viruses in cold adapted live attenuated influenza vaccines met the requirement for both conventional and emergency inspection on cold adapted live attenuated influenza vaccines.
