Specific and Sensitive Detection of H. pylori in Biological Specimens by Real-Time RT-PCR and In Situ Hybridization

Abstract

PCR detection of H. pylori in biological specimens is rendered difficult by the extensive polymorphism of H. pylori genes and the suppressed expression of some genes in many strains. The goal of the present study was to (1) define a domain of the 16S rRNA sequence that is both highly conserved among H. pylori strains and also specific to the species, and (2) to develop and validate specific and sensitive molecular methods for the detection of H. pylori. We used a combination of in silico and molecular approaches to achieve sensitive and specific detection of H. pylori in biologic media. We sequenced two isolates from patients living in different continents and demonstrated that a 546-bp domain of the H. pylori 16S rRNA sequence was conserved in those strains and in published sequences. Within this conserved sequence, we defined a 229-bp domain that is 100% homologous in most H. pylori strains available in GenBank and also is specific for H. pylori. This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes. The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys. This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens.

Antibiotic Treatment Decreases Microbial Burden Associated with Pseudomyxoma Peritonei and Affects β-Catenin Distribution

“…26). In initial experiments, we used both cRNA and cDNA probes concurrently, and confirmed earlier observations that these 2 probes can detect the same bacteria (27).…”

Comparative Evaluation of RUT, PCR and ELISA Tests for Detection of Infection with Cytotoxigenic H. pylori

“…These findings were consistent with results of previously reported studies. 30,31 Low Sensitivity of PCR is possibly due the presence of inhibitors of the polymerase enzyme which adversely impact the outcomes. 16 Besides, as a passive test, distinction between live and dead organisms is not possible via PCR and it might results in false positive.…”

Mutations to Essential Orphan Response Regulator HP1043 of Helicobacter pylori Result in Growth-Stage Regulatory Defects

“…Quantitative RT-PCR (qRT-PCR) of the target genes was performed for each of the cDNA reactions in a CFX96 real-time system (Bio-Rad Laboratories, CA) with GoTaq quantitative PCR (qPCR) master mix (Promega, WI). The H. pylori 16S rRNA gene was used as the internal control for qRT-PCR (26). The reaction mixtures were incubated for 10 min at 95°C to activate the GoTaq DNA polymerase, followed by 35 amplification cycles of 95°C for 15 s, 55°C for 15 s (optimized by gradient PCR of genomic DNA), and Bio-Rad software was used to perform statistical analyses of the data acquired from the qRT-PCR.…”