Abstract
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. J. Clin. Lab. Anal. 11:323–327, 1997. © 1997 Wiley‐Liss, Inc.
Keywords: RT‐PCR, uracil N glycosylase, thermus thermophilus (Tth), DNA polymerase
References
- 1. Cherian T, Bobo L, Steinhoff MC, Karron RA, Yolken RH: Use of PCR‐enzyme immunoassay for identification of influenza A matrix RNA in clinical samples negative for cultivable virus. J Clin Microbiol 32: 623–628, 1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Mulder J, McKinney N, Christopherson C, Sninsky J, Greenfield L, Kwok S: A rapid and simple PCR assay for quantitation of HIVRNA: Application of acute retroviral infection. J Clin Microbiol 32: 292–300, 1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Wright KE, Wilson GAR, Novosad D, Dimock C, Tan D, Weber JM: Typing and subtyping of influenza viruses in clinical samples by PCR. J Clin Microbiol 33: 1180–1184, 1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Kawasaki ES: Amplification of RNA In PCR Protocols: A Guide to Methods and Applications. Innis MA, Gelfend DH, Sninsky JJ, White TJ, eds. Academic Press, San Diego, 1990, p 21–27. [Google Scholar]
- 5. Myers TW, Gelfand DH: Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry 30: 7661–7666, 1991. [DOI] [PubMed] [Google Scholar]
- 6. Young KKY, Resnick RM, Myers TW: Detection of hepatitis C virus RNA by combined reverse transcription‐polymerase chain reaction assay. J Clin Microbiol 31: 882–886, 1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Longo MC, Berninger MS, Hartley JL: Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions. Gene 93: 125–128, 1990. [DOI] [PubMed] [Google Scholar]
- 8. Slupphaug G, Alseth I, Eftedal I, Volden G, Krokan HE: Low incorporation of dUMP by some thermostable DNA polymerases may limit their use in PCR amplifications. Anal Biochem 211: 164–169, 1993. [DOI] [PubMed] [Google Scholar]
- 9. Myers TW, Sigua CL: Amplification of RNA: High‐temperature reverse transcription and DNA amplification with Thermus thermophilus DNA polymerase In: PCR Strategies. Academic Press, New York, 1995, p 58–68. [Google Scholar]
- 10. Mateo R, Faruki H, Copper DL, Demetris AJ, Enrlich GD: Detection of hepatitis C virus RNA in liver and plasma by reverse transcriptase PCR using liquid hybridization In: PCR‐based Diagnostics in Infectious Disease. Enrlich GD, Greenberg SJ, eds. Blackwell, Boston, 1994, p 375–397. [Google Scholar]
- 11. Sambrook J, Fritsch EF, Maniatis T: In: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [Google Scholar]
- 12. Thornton CG, Hartley JL, Rashtchian A: Utilizing uracil DNA glycosylase to control carry over contamination in PCR: Characterization of residual UDG activity following thermal cycling. BioTechnique 13: 180–184, 1992. [PubMed] [Google Scholar]