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The Inhibitory Effect of Mouse Gastric DNA on Amplification of Helicobacter pylori Genomic DNA in Quantitative PCR
Mehdi Alikhani , Ebrahim Shafaie , Esmat Mirabzadeh Ardakani , Maryam Esmaeili , Samaneh Saberi , Mojgan Hatefi , Marjan Mohammadi *
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
Full-Text [PDF 663 kb]      
Type of Study: Short Communication | Subject: Related Fields
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