Optimized PCR amplification of influenza A virus RNA using Tth DNA polymerase, incorporating uracil N glycosylase (UNG) in a single tube reaction.
An optimized reaction condition for amplification of influenza A virus RNA, by thermus thermophilus (Tth) DNA polymerase-based PCR, incorporating uracil N glycosylase (UNG) and dUTP in the reaction has been determined. dUTP could not be substituted for all dTTP sites when UNG was present in the reaction. The relative concentration of dUTP and dTTP has been optimized for allowing amplification of the target RNA. It has been verified that the amplified product DNA had sufficient dUTP and was digestable by UNG. Using the optimized reaction condition, influenza A virus-specific DNA fragment could be amplified and detected in 15 of 15 culture positive (for influenza A virus) nasopharyngeal specimens.
Poddar
S K
SK
Department of Pediatrics, University of California at San Diego, La Jolla, California.
Sawyer
M H
MH
Connor
J D
JD
D045647:DNA Glycosylases; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D006801:Humans; D009980:Influenza A virus / Q000235:genetics*; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009305:Nasopharynx / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D016963:Thermus thermophilus / Q000201:enzymology*; D013942:Thymine Nucleotides / Q000378:metabolism; D051981:Uracil-DNA Glycosidase
D003856:Deoxyuracil Nucleotides; D012367:RNA, Viral; D013942:Thymine Nucleotides; C027078:deoxyuridine triphosphate; D004259:DNA-Directed DNA Polymerase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; C024157:thymidine 5'-triphosphate