The Research Of A Kind Of Method For Rapid Detection Of Campylobacter Jejuni

Campylobacter jejuni is now the main cause of bacterial diarrheain the developed countries. As a kind of food-borne pathogen, cam-pylobacter jejuni now becomes a significant challenge to public he-alth. Poultry has become the major source of protein in most Chi-nese people's lives, including milk and eggs. The number of humancampylobacteriosis has become more recently. Therefore the researchshould be carried out in order to detect campylobacter jejuni muchmore quickly and sensitively, because of the weakness of the traditional detective methods.Objective: To Set up a multiplexed PCR method for rapid detection ofCampylobacter jejuni from food. Then it will be used for detectingcampylobacter jejuni in normal work. Then we will know the state of presence of campylobacter jejuni in Nanjing.Methods: Based on the sequence of hipO, mapA and 23S rRNA, whichwere recorded in gene bank about campylobacter jejuni, several couplesof specific primers were designed by two kinds of primer-design software(Primer Premier5 and Oligo 6). The most specific primers pairs werepicked out. And the optimization of the concentration of PCR, such asprimer pairs, Mg²⁺ and Tm℃were also found out.Results: 1. This method of multiplex PCR has confirmed its highspecificity by several experiments, in which dozens of standard in-testinal pathogenic bacteria strains were used as the templates, inclu-ding campylobacter jejuni, campylobacter coli, Escherichia coli, andsaimonella, etc. Only when campylobacter jejuni was used as thetemplate could three specific fragments was found in a time, whileone fragment was got when campylobacter coli be used. Nothingwas detected when other be used intestinal pathogenic bacteria.2. Different concentration of campylobacter jejuni(from 10^-4 to 10^-3)was used as the PCR Templates. The results showed that this sensi-tivity level for this method was about 15 CFU. Meanwhile the sa-me cultures were incubated for 48 hours at 42℃in microaerobic atmosphere and then counted the colonies. The result confirmed thatthe sensitivity for traditional method was 45 CFU, which was lowerthan the multiplex PCR. 3. About 290 portions of poultry samples were collected from several markets in Nanjing from September 2005 to June 2006. Each sa-mple was detected both with the traditional method and the multi-plex PCR. In all 48 positive strains (16.6%) were detected by thetraditional method. And 110 positive strains (37.9%) were detectedby the multiplex PCR method, which was much more than the tra-ditional method.Conclusion: Campylobacter jejuni can be detected by the multiplexedPCR method from many kinds of food, whether fresh, chilled or frozen. Itis saving of time, much convenient and highly efficient, and can be easilyutilized in normal work. After making use of the multiplexed PCRmethod for several months, the prevalence of campylobacter jejuni inNanjing is about 37.9%.