The Inhibitory Effect of Mouse Gastric DNA on Amplification of Helicobacter pylori Genomic DNA in Quantitative PCR

Authors
Mehdi Alikhani 1
Ebrahim Shafaie 1
Esmat Mirabzadeh Ardakani 2
Maryam Esmaeili 1
Samaneh Saberi 1
Mojgan Hatefi 1
Marjan Mohammadi 1
1 HPGC Research Group, Medical Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran
2 Animal Sciences Lab, Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran
10.29252/ibj.23.4.297
Abstract
Background: Quantitation of Helicobacter pylori (Hp) in the gastric tissue is essential for assessment of vaccination/therapeutic regimens. Methods & Results: Here, the inhibitory effect of mouse gastric DNA (MgDNA) on amplification of Hp genomic DNA (HpDNA) was evaluated by spiking HpDNA with serial dilutions of MgDNA, which yielded concentrations of >10 ng/µl and >0.63-10 ng/µl of MgDNA, as inhibition and interference zones, respectively. Mice were then inoculated with varying doses of Hp and assessed at the inhibition-free concentration of 0.63 ng/µl. The average Hp copy numbers per microgram of gastric tissue discriminated mice having received high vs. low dose inoculums (p < 0.001). Secondly, Hp copy numbers were quantitated in immunized mice, which demonstrated significantly lower numbers, in reference to controls (p = 0.006). Conclusion: Our method, bypassing the inhibition and/or interference imposed by MgDNA, was able to quantitate gastric tissue-colonizing Hp, segregating mice inoculated with low vs. high doses of Hp, as well as those immunized from controls.
Keywords
Amplification
Inhibition
interference
Standard curve
Quantitative PCR