Effect of inhibitors in clinical specimens on Taq and Tth DNA polymerase-based PCR amplification of influenza A virus.
Fifteen randomly selected nasopharyngeal (NP) swab specimens (culture-negative for influenza A virus) were spiked with influenza A virus and the nucleic acids were extracted and subjected to PCR amplification with Thermus aquaticus (Taq) and T. thermophilus (Tth) DNA polymerases. Products of the expected size, and giving equivalent band intensities, were obtained from four specimens with both polymerases. Fox six specimens, less products were obtained with Taq DNA polymerase than with Tth DNA polymerase. Products were detected from five NPs only by PCR with Tth DNA polymerase. The transport medium and the calcium alginate swab fibre of the specimens were shown not to be the source of the inhibitors. The incorporation of 32P-dCTP into cDNA, and the yield of PCR products of cDNA made from control RNA template (purified from H2O spiked virus suspension) were decreased in the presence of inhibitory extracts, showing that both the reverse transcription (RT) and PCR steps in amplification with Taq DNA polymerase were sensitive to the inhibitors. In contrast, Tth DNA polymerase was more resistant to the inhibitors and viral nucleic acid from all the specimens examined could be amplified and detected in a single step by RT-PCR with Tth DNA polymerase.
Poddar
S K
SK
Department of Pediatrics, University of California at San Diego, La Jolla 92093-0808, USA.
Sawyer
M H
MH
Connor
J D
JD
D018076:DNA, Complementary / Q000032:analysis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000302:isolation & purification; D009305:Nasopharynx / Q000821:virology*; D019384:Nucleic Acid Synthesis Inhibitors; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D019914:Taq Polymerase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013824:Thermus / Q000201:enzymology
D018076:DNA, Complementary; D019384:Nucleic Acid Synthesis Inhibitors; D012367:RNA, Viral; D019914:Taq Polymerase; C068508:Tth polymerase; D004259:DNA-Directed DNA Polymerase