sybr-green-i and Helicobacter-Infections

The feasibility of using a polymerase chain reaction (PCR)-based label-free DNA sensor for the detection of Helicobacter pylori is investigated. In particular, H. pylori ureC gene, a specific H. pylori nucleic acid sequence, was selected as the target sequence. In the presence of ureC gene, the target DNA could be amplified to dsDNA with much higher detectable levels. After added the SYBR green I (SGI), the sensing system could show high fluorescence. Thus, the target DNA can be detected by monitoring the change of fluorescence intensity of sensing system. The clinical performance of this method was determined by comparing it with another conventional technique urea breath test (UBT). The result also showed good distinguishing ability between negative and positive patient, which was in good agreement with that obtained by the UBT. It suggests that the label-free fluorescence-based method is more suitable for infection confirmation test of H. pylori. This approach offers great potential for simple, sensitive and cost-effective identification of H. pylori infection.

Topics: Benzothiazoles; Breath Tests; Diamines; DNA; Fluorescent Dyes; Genes, Bacterial; Helicobacter Infections; Helicobacter pylori; Humans; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Sensitivity and Specificity; Spectrometry, Fluorescence

Accurate diagnosis of Helicobacter pylori infection is important in both clinical practice and clinical research. Molecular methods are highly specific and sensitive, and various PCR-based tests have been developed to detect H. pylori in gastric biopsy specimens. We optimized a sensitive and specific quantitative SYBR Green I real-time PCR assay for detection of H. pylori based on amplification of the fragment of a 26-kDa Helicobacter species-specific antigen gene that allows for detection of 5 bacterial cells per PCR sample. Under the assay conditions, SYBR Green I real-time PCR is highly reproducible with a precise log-linear relation in the range of six orders of magnitude of bacterial DNA concentrations. For accurate comparison of H. pylori infection in different tissue samples, the amount of total host DNA in each sample is normalized by TaqMan real-time PCR of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pseudogenes. The developed method was validated in prophilactically immunized and experimentally infected mice and revealed a level of H. pylori gastric colonisation that was below the limit of detection for a rapid urease test. This new method established for a quantitative analysis of H. pylori in the host's stomach may be useful in experimental studies evaluating new anti-H. pylori drugs and vaccines.

Topics: Animals; Antigens, Bacterial; Benzothiazoles; Diamines; DNA, Bacterial; Gastric Mucosa; Helicobacter Infections; Helicobacter pylori; Mice; Mice, Inbred C57BL; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Sensitivity and Specificity; Specific Pathogen-Free Organisms; Stomach Diseases; Urease