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, r Tth had the lowest sensitivity to bile inhibitors, whereas Taq and Tfl had the highest sensitivity. Bile proteins did not inhibit Ampli Taq DNA polymerase, whereas the fraction containing mainly bile acids and their salts inhibited the amplification capacity of Ampli Taq . Heating human bile at 98 °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 r Tth DNA polymerase was found efficient to amplify DNA directly in bile.