| Campylobacter jejuni(abbreviated as C.jejuni) is a food-borne pathogen that can cause diseases to both people and animals all around the world and it has been extensively attractive to researchers during the last two decades.C.jejuni is one species out of Campylobacter genus,and it is believed to be one of the major pathogens causing human bacterial enteritis.Clinical manifestation of campylobacteriosis may generally be summarized as enteritis and/or infection outside intestines.At present,clinical detection of C.jejuni mainly depends on culture methods,including direct isolation,pre-enrichment culture and filtration method,all of which are time-consuming and laborious.Suspected colonies are needed to be confirmed by lots of biochemical tests in the end.Therefore,the result will come out in more than 5-7 days,which may delay the diagnosis and corresponding therapy. Due to the disadvantages above,many scientists have developed methods of molecular biology and immunology to detect C.jejuni,including common PCR, oligonucleotide probe,ELISA,immunomagnetic beads and so on.However,all of these methods are confined to pure cultures of C.jejuni and foods or water for rapid screening,which are not commonly applied to detect C.jejuni in human diarrheal stool samples.Hence,in this study,we want to carry out real-time PCR as an alternative to culture methods for rapid and accurate detection of C.jejuni in human diarrheal stool.Objective:To establish a quick and accurate real-time PCR method to detect C. jejuni in human diarrheal stool as an alternative to traditional culture methods.Materials and Methods:1.256 stool samples were collected from inpatients and outpatients with the syndrome of diarrhea.Additionally,50 stool samples were collected from healthy people.2.All stool samples were detected by the filtration method using 3 culture media to ensure the reliability of the results,which included C.jejuni selective blood agar, Columbia blood agar and CCDA plate.3.The real-time PCR,using the primer-probe particularly designed for the specific sequence of ORF-C gene of C.jejuni,was applied to detect C.jejuni(5 strains), other species of Campylobacter(6 types) and other enteric bacteria(17 types) for specificity.Sensitivity test for the primer-probe was performed through serial dilution of pure cultures of C.jejuni and artificially contaminated stool samples with C.jejuni.4.Bacterial genome DNA was extracted from 306 stool samples homogenized with diluting solution for real-time PCR and then the results would be compared with that of culture methods.5.During the whole process of the study,the results of culture methods and real-time PCR were double-blinded.Results:1.Of 256 diarrheal stool specimens,the number of positive ones for C.jejuni selective blood agar,Columbia blood agar,and CCDA plate was 10,10 and 9, respectively.Of the 50 healthy stool samples,no C.jejuni was detected by the 3 culture media.The concentration of C.jejuni in positive stool samples was about 10~4-10~6CFU/g. 2.Real-time PCR with the particular primer-probe was found to be specific to C. jejuni and the lowest detection limit in pure cultures and stool samples was about 10~2 CFU/mL and 10~3 CFU/g,respectively.3.Of the 256 diarrheal stool samples,the same 10 positive samples were successfully detected by real-time PCR.In addition,2 other samples were also found to be positive by real-time PCR,which were confirmed to be C jejuni by sequencing. No positive result was found from 50 healthy stool specimens.4.Taking culture method as the golden standard,the specificity and sensitivity of real-time PCR was 99.2%and 100%respectively and the Youden's index was 0.992.The difference between the 2 methods was not significant by McNemar test and the Kappa value was 0.905.Conclusion:1.The primer-probe was highly specific to C.jejuni and its lowest detection limit was suitable for clinical tests.2.Real-time PCR with reliable results is convenient and time-saving,so that it is suitable for detecting C.jejuni in stool samples.
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