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
Iranian Biomedical Journal
Volume 23, Issue 4 - Serial Number 86
July and August 2019
Pages 297-302