Characterization of the PCR inhibitory effect of bile to optimize real-time PCR detection of Helicobacter species.
The inhibitory effect of human and porcine bile samples to detect Helicobacter DNA was studied by adding different concentrations of bile samples to PCR mixtures of six thermostable DNA polymerases containing cagA specific primers and Helicobacter pylori DNA. PCR products were amplified by using the Rotorgene system and SYBR Green I. Among the six DNA polymerases tested, rTth had the lowest sensitivity to bile inhibitors, whereas Taq and Tfl had the highest sensitivity. Bile proteins did not inhibit AmpliTaq DNA polymerase, whereas the fraction containing mainly bile acids and their salts inhibited the amplification capacity of AmpliTaq. Heating human bile at 98 degrees C and adding casein and formamide to the reaction mixture reduced the PCR inhibitory effect of bile. Therefore, a pre-PCR treatment based on dilution and heating of bile, adding casein and formamide to the reaction mixture of rTth DNA polymerase was found efficient to amplify DNA directly in bile.
Al-Soud
Waleed Abu
WA
Department of Medical Microbiology, Dermatology and Infection, Lund University, Sölvegatan 23, SE-223 62 Lund, Sweden. abu.al-soud@mmb.lu.se
Ouis
Ibn-Sina
IS
Li
Dai-Qing
DQ
Ljungh
Sa
S
Wadström
Torkel
T
D000818:Animals; D001646:Bile / Q000737:chemistry*; D001647:Bile Acids and Salts / Q000494:pharmacology*; D004795:Enzyme Stability; D016998:Helicobacter / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006358:Hot Temperature; D006801:Humans; D008297:Male; D051379:Mice; D009077:Mucins / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors; D016133:Polymerase Chain Reaction / Q000379:methods*; D013552:Swine
D001647:Bile Acids and Salts; D009077:Mucins; D019384:Nucleic Acid Synthesis Inhibitors