Detection Of Campylobacter Jejuni And Helicobacter Pylori By Loop-mediated Isothermal Amplification Method

ObjectiveThe aim of this study is to establish a loop-mediated isothermal amplification method for detection (LAMP) of Campylobacter jejuni and Helicobacter plyori through turbidity meter and colorimetric method and then verify the specificity and sensitivity of the method.Methods1. Five to six sets of primer were designed to recognize the specific gene, HipO gene of C.jejuni and UreB gene of H. pylori.2. Several sets of primers were selected by high amplification efficiency for C. jejuni and H. pylori.3. The specificity of primers was tested with the control of other 24 bacterial strains and ddH2O. The optimal set of primers was determined by amplification efficiency and specificity.4. Then the optimal temperature of this reaction was selected from 58-65?.5. The sensitivity was respectively examined using serial dilutions of C. jejuni and H.pylori DNA as templates.6. Stool samples and gastric juice samples were collected to compare the positive rate,sensitivity and specificity of LAMP with that of other conventional methods.Results1. The optimal primer combination for LAMP of C. jejuni was CJ-5 and which of H.pylori was HPUB-3. All reactions were finished at a constant temperature in 60 minutes.2. Detection of C. jejuni and H. pylori by LAMP both had good specificity.3. The results recording by turbidity meter showed that the sensitivity of LAMP detecting C. jejuni and H. pylori LAMP with a detection limit of 6.97×102copies/?l and 2.61×103copies/?l, respectively.4. C. jejuni was not found by LAMP, PCR and culture method in 50 clinical samples. The positive rate, sensitivity and specificity of H. pylori detecting by LAMP was 41.25%,94.3% and 95.7%. The results were no significant difference between LAMP and other methods, such as pathological examination and 13C urea breath test (P>0.05).Conclusion1. In this study, LAMP primers were designed targeting HipO gene and UreB gene to detect C. jejuni and H. pylori respectively, which had high amplification efficiency and good specificity.2. The results of clinical gastric juice test showed that the H. pylori positive rate detecting by LAMP had no significant difference with other conditional methods and had high sensitivity and specificity.3. LAMP is a potential and valuable method of detection of C. jejuni and H.pylori due to its rapidity, simplicity, low cost and accuracy. It is especially suitable for primary medical units.