| Campylobacter jejuni(C.jejuni) and Campylobacter coli(C.coli) are major thermcampylobacter, they can cause many diseases of human and animals. They are stuff-original pathogenic bacteria. To a certain extent, their infection exceeds Salmonella's. C.jejuni and C.coli have been isolated frequently in diarrhea patients, and they are threatening the safe of poultry product and human health. At present, thermcampylobacter has been one of the chief pathogenic bacteria detected in poultry products of export and import in China.In the inspection and quarantine of export and import for animal products, the inspection of sample's pathogenic bacteria are undertaking working standards—GB and SN. However, some traditional methods exists inevitably the problems of complicated operation, many steps, long cycle, etc. So it can't be satisfied with the need of gross sample's inspection and quarantine. They become restrictive factors for export and import of animal products.The study established the methods of PCR and nucleic acid hybridization to detect the thermcampylobacter in China. The methods simplified inspection flow-sheet, enhanced detective efficiency and can be applied in practical sample's detection. 16SrRNA of C.jejuni and C.coli with high homology was selected to amplify target gene and designed primers by using the software of Primer 5.0. The PCR method to detect C.jejuni and C.coli has been established in chicken samples. The cycling program was 94℃ for 4 min followed by 25 cycles of 94℃ for 30 s, 58℃ for 30 s, and 72℃ for 1min and a final extension step at 72℃ for 4 min. The optimized composition of PCR was selected as that template was diluted 10-3, the density of primer was 0.4umol/L. The 287bp nucleotide fragment of C.jejuni or C.coli can be amplified specifically by PCR, but can't be amplified from C.fetus, Staphylococcus aureus, Salmonella enteritidis, Clostridium perfringens, V.parahaemolyticus, B.cereus, Aeruginosus Bacillus, L.monocytogenes and so on. The sensitivity of PCR to detect the bacteria was 5 cfu/mL. On the basis of established PCR method, the nucleic acid probe was designed and synthetized according to the 16SrRNA of C.jejuni and C.coli. The length of probe is 21bp. This method utilized a chemiluminescent acridinium ester to label an oligonucleotide probe that was designed to hybridize with a target oligonucleotide sequence. And then, they generated DNA-RNA hybridity. After hybridization, hybridized probe and unhybridized probe can be identified without separation, but by differential hydrolysis, that was, when adding mild alkaline, unhybridizied probe lost luminous character (the half-life period is less than 1 minute), but the solution hybridization of the probe with the target sequence protected the acridinium ester from mild alkiline hydrolysis. Because the acridinium ester with plane structure was set in double-helical interior and protected by stereospecific blockade, the speed of hybridized probe hydrolysis was lowed down (the half-life period is more than 10 minute), so that the acridinium ester still had luminous efficiency. Therefore, when adding alkaline hydrogen peroxide, only the chemiluminescence of the hybridized probe can be detected in a luminometer. 30 minutes could be used during the process. This method is named nucleic acid hybridization protection assay (HPA), but it was applied less in China now.The pure culture's colonies of C.jejuni type strain were detected by HPA. C.jejuni and C.coli can be detected specifically by HPA at screening and optimizing reactive condition, but the...
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