Rapid Detection of Campylobacter jejuni by LAMP (Loop_mediated isothermal DNA amplification) in poultry meat

Abstract

Objective: Food born Disease are of most important health problems worldwide and the genus Campylobacter is a crucial threaten to public health because it includes several species that may cause diarrhea. On the other hand, detection is problematic due to microaerophilic nature of the bacteria. Aim: The objective of present thesis is to evaluate a developed LAMP method as an alternative to conventional and PCR method in detection of C. jejuni. Methods: Phenotypical confirmation of C. jejuni was achieved by culturing the samples in campylobacter selective agar base medium, gram staining, oxidase and catalase tests. The DNA was extracted by standard method. The HipO gene was designated and amplified using six sets of primers for LAMP in single temperature in water bath (58℃).Also detection of C. jejuni was confirmed by the outer primers of LAMP in PCR. The amplified products were detected by gel electrophoresis and visualized by their turbidity with naked eye as well as Fluorescent substance under transilluminator. Results: C. jejuni detection was confirmed in Campylobacter Selective Agar Base medium by producing gray colonies followed by purple dye in oxidase test and bubbles in catalase test. The PCR generated several PCR products with a length of 300 bp until 800 bp, for the C. jejuni by the outer primers of LAMP. The LAMP detection method products were observed with the naked eye as the opacity of these products increased, and it was identified by using fluorescent materials. To confirm the result, it was detected by gel electrophoresis, which represents the ladder-shaped products. Conclusion: According to the result, it was shown that Conventional PCR method for detection of Campylobacter needs standard thermoscycler and takes 3 hours, but by using LAMP method, we were able to amplify and detect Campylobacter in simple water bath, in much less time (within 60 minutes).