Quantitative detection for low levels of Helicobacter pylori infection in experimentally infected mice by real-time PCR.
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.
Mikula
Michał
M
Department of Gastroenterology, Medical Center for Postgraduate Education, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, ul. Roentgena 5, 02-781, Warsaw, Poland.
Dzwonek
Artur
A
Jagusztyn-Krynicka
Katarzyna
K
Ostrowski
Jerzy
J
D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics; D052160:Benzothiazoles; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D003959:Diamines; D005753:Gastric Mucosa / Q000382:microbiology; D016481:Helicobacter Infections / Q000175:diagnosis* / Q000382:microbiology; D016480:Helicobacter pylori / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D008810:Mice, Inbred C57BL; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D013047:Specific Pathogen-Free Organisms; D013272:Stomach Diseases / Q000175:diagnosis* / Q000382:microbiology; D014510:Urease / Q000737:chemistry / Q000235:genetics
D000942:Antigens, Bacterial; D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D014510:Urease