Microfluidic analysis of multiplex PCR products for the genotyping of Helicobacter pylori Application
نویسندگان
چکیده
Gastric infection with Helicobacter pylori (H. pylori) is the major cause of chronic active gastritis and is associated with the pathogenesis of peptic ulcer and gastric carcinoma. Recent studies using a multiplex PCR (mPCR) based approach distinguished different allelic variants that are associated with different stages of H. pylori virulence. H. pylori genotyping on gastric tissues, routinely processed in diagnostic pathology, revealed that certain combinations of virulence subtypes of different H. pylori sub-strains are associated with gastric carcinoma and MALT lymphoma. The use of formalin fixed samples as the source for template DNA for mPCR restricts the size of the PCR fragments to 300 bp, due to poor DNA quality. For the analysis of multiple bands of a mPCR experiment, the data acquisition from slab gels is often inconvenient in terms of sizing, resolution, and data accuracy. To improve accuracy and reproducibility of the measurements, Mascha B. Mues, Christoph I. Koehler and Margarete Odenthal the Agilent 2100 bioanalyzer using Lab-on-a-chip technology was tested to replace slab gels as the tool for the electrophoretic separation of the mPCR products. First analyses revealed an improved fragment resolution, sizing accuracy and reproducibility. A mPCR reaction covering five different alleles that are involved in H. pylori pathogenesis was analyzed and all fragments were separated and quantified. The analysis of a mixture of all mPCR reactions including all involved alleles showed the separation of seven different PCR fragments in the range of 102 to 301 bp, thereby expanding the spectrum of prognostic or therapeutically relevant information used e.g. in H. pylori diagnostic.
منابع مشابه
Simultaneous Detection of Helicobacter Genus and Helicobacter pylori Species using a Multiplex PCR Method
In order to improve simultaneous detection and identification of Helicobacter genus in general and Helicobacter pylori specifically and reduce the number of amplifications needed, we established a multiplex PCR. In this study, two pairs of primers: Hcom1 and Hcom2 specific for Helicobacter genus, Hicd1 and Hicd2 specific for Helicobacter pylori species were used. To determine the sensitivity of...
متن کاملVacA and cagA genotypes status and antimicrobial resistance properties of Helicobacter pylori strains isolated from meat products in Isfahan province, Iran
Although Helicobacter pylori has a significant impact on the occurrence of severe clinical syndromes, its exact ways of transmission and origin have not been identified. According to the results of some previously published articles, foods with animal origins play a substantial role in the transmission of H. pylori to humans. The present investigation was carried out to study the vacuolating cy...
متن کاملMultiplex PCR assay for rapid detection and genotyping of Helicobacter pylori directly from biopsy specimens.
We developed and evaluated a simple, novel multiplex PCR assay for rapid detection of Helicobacter pylori infection and for the determination of vacA and cagA genotypes directly from gastric biopsy specimens. This assay did not require culturing of strains or extraction of DNA from biopsy samples. This multiplex PCR assay would be of particularly great value for laboratories in developing count...
متن کاملPrevalence of Coinfection Helicobacter pylori with Non-Helicobacter pylori Helicobacters Species in Patients Suffering from Gastric Diseases in Iran
Introduction: In addition to Helicobacter pylori, non-Helicobacter pylori helicobacters (NHPHs) have been diagnosed in the humans stomach that caused gastrointestinal diseases. This study aimed to evaluate the coinfection of H. pylori with NHPHs species in patients with gastric disorders in Iran. Materials & Methods: This cross-sectional study was performed on 421 gastric biopsies form dysp...
متن کامل