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Microfluidic analysis of multiplex,,,PCR products for the genotyping of,,,Helicobacter pylori

e and 50 % isopropanol.
PCR
For H. pylori genotyping, a sequence specific, nested PCR approach was performed as described earlier by Koehler et al. [5]. Briefly, genomic sub-fragments of the virulence factors vacA, cagA, and iceA were amplified by the combined use of allelespecific primer sets (vacA s1/s2, vacA m1/m2, cagA, and iceA 1/2) in two PCR amplification steps, each starting with a preincubation at 95C for 5 minutes, then followed by 35 cycles of 1 minute denaturation at 95 C, 1 minute annealing at 58 C and 1 minute extension at 72 C. PCR yielded products with sizes varying from 102 bp to 301 bp.
Analysis of PCR-products
PCR products were analyzed using standard agarose gel electrophoresis and Lab-on-a-chip technology on the Agilent 2100 bioanalyzer using the Agilent DNA 1000 LabChip kit according to the manufacturer's instructions.
Results and discussion
In a previous study 5 we have established a nested multiplex PCR (mPCR) assay in order to identify the different virulence factors of Helicobacter pylori, vacA, cagA and iceA genotypes in routinely processed gastric biopsies which are formalin fixed and paraffin embedded. The nested mPCR assays with a limited number of primer sets produced reliable, distinct amplification products of all specimens. The combined PCR assays for the simultaneous detection of the vacA midregions m1 and m2, as well as the combined detection of iceA1 and iceA2 were routinely performed. The semi-nested PCR for the detection of the vacA signal regions s1 and s2 was usually combined with the nested PCR for the cagA gene. The established mPCR assay permits the simultaneous analysis of all seven different diagnostically relevant alleles. However, tested on a broad spectrum of di
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