Develonment and application of TaqMan MGB probe real-time fluorescence quantitative PCR for rapid detection of Helicobacter hepaticus
10.3760/cma.j.issn.0254-5101.2011.09.020
- VernacularTitle:肝螺杆菌TaqMan MGB探针实时荧光定量PCR快速检测方法的建立及应用研究
- Author:
Zhengqin GAO
;
Jin XING
;
Yufang FENG
;
Bingfei YUE
;
Zhengming HE
- Publication Type:Journal Article
- Keywords:
Helicobacter hepaticus;
Minor groove binder (MGB) probe;
Real-time fluorescence quantitative PCR;
Detection
- From:
Chinese Journal of Microbiology and Immunology
2011;31(9):833-838
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a TaqMan MGB probe-based,sensitive and specific real-time fluorescence quantitative PCR assay for rapid detection of Helicobacter hepaticus.Methods Primers and probes specific toflaB gene of Helicobacter hepaticus were designed.A TaqMan MGB probe-based,real-time fluorescence quantitative PCR was established.The specificity,sensitivity and stability of the assay were assassed.Then,the established TaqMan MGB probe real-time fluorescence quantitative PCR assay was applied to detect Helicobacter hepaticus in 1081 clinical specimens during 2008-2011,compared with bacterial isolation and culture method and conventional PCR assay.Results The specificity of this established TaqMan MGB probe-based real-time fluorescence quantitative PCR was high and there were no cross-reactivity with Helicobacter pylori,Campylobacter jejuni,Clostridium piliforme,Pasteurella pneumotropica,Escherichia coli,Pseudomonas aeruginosa.The detection limits was 8.3 copies.The correlation coefficient and slope value of standard curve were 0.999 and -3.227,respectively and the efficiency of TaqMan MGB-based probe realtime fluorescence quantitative PCR assay was 100％.The TaqMan MGB-based probe real-time fluorescence quantitative PCR and conventional PCR were preformed to detect Helicobacter hepaticus in 1081 clinical specimens,a total of 86 specimens were positive for Helicobacter hepaticus.However,there was only 4 specimens were positive by bacteria isolation and culture method.The results showed that TaqMan MGB -based probe real-time fluorescence quantitative PCR for Helicobacter hepaticas was more sensitive than bacteria isolation and culture method,and it could detect Helicobacter hepaticus DNA from clinical specimens directly,and detection time is only 2 hours.Conclusion The TaqMan MGB-based probe real-time fluorescence quantitative PCR assay was a reliable,specific,sensitive and useful tool for rapid detection of Helicobacter hepaticus.
