A multiplex RT-PCR method for screening of reassortant live influenza vaccine virus strains.
A system based on reverse transcription polymerase chain reaction (RT-PCR) of the RNA genome was established to identify genetic composition of influenza viruses generated by reassortment between an attenuated donor virus and virulent wild type virus. The primers were designed, by multiple sequence alignment of variable regions, specific for cold-adapted donor virus HTCA-A 101, as compared to other influenza A viruses. The specificity of each primer set was confirmed and the primers were combined to perform RT-PCR in multiplex manner. The multiplex PCR was adopted to distinguish the 6:2 reassortant viruses containing six internal genome segments of attenuated donor virus and two surface antigens of virulent strain from the wild type viruses. The method allowed us to optimize the reassorting process on a routine basis and to confirm the selection of reassortant clones efficiently. The method is suitable for analyzing the contribution of specific gene segments for growth and attenuating characteristics and for generation of live attenuated vaccine by annual reassortment.
Ha
Suk-Hoon
SH
Department of Biotechnology, College of Engineering, Yonsei University, 134 Shinchon-Dong, Seodaemun-Gu, Seoul 120-749, South Korea.
Kim
Hyun Ah
HA
Kim
Yeon Hee
YH
Kim
Jae Seoung
JS
Lee
Kwang Hee
KH
Park
Song Yong
SY
Park
Wan-Je
WJ
Seong
Baik Lin
BL
D000222:Adaptation, Physiological; D000818:Animals; D002642:Chick Embryo; D003080:Cold Temperature; D017931:DNA Primers; D018076:DNA, Complementary; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics / Q000502:physiology; D009980:Influenza A virus / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D016865:Reassortant Viruses / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral; D014759:Viral Envelope Proteins