| 摘要: |
| 【目的】建立一种特异、灵敏的牛源幽门螺杆菌(Helicobacter pylori，Hp)检测方法，为家畜感染幽门螺杆菌的流行病学调查鉴定提供支持。【方法】分别选取Hp的16S rRNA、UreA、glmM为靶基因设计特异性引物，以H.pylori动物模型适应菌株SS1株为标准菌株建立PCR检测方法，并进行PCR条件优化，运用所建立的最优PCR方法检测临床奶牛乳样和粪样中Hp的分布情况。【结果】PCR特异性试验结果显示，仅Hp SS1株能扩增出特异性条带，而对照菌株金黄色葡萄球菌、沙门氏菌、单核细胞增生李斯特氏菌、大肠杆菌及蜡样芽孢杆菌均无扩增条带。PCR灵敏度试验结果显示，16S rRNA、UreA、glmM基因在乳样中的最低检出浓度分别为101，103和103 CFU/mL，在粪样中的最低检出浓度分别为101，104和104 CFU/mL。应用该PCR方法对采集自规模化养殖场及散养的共计41头奶牛的乳样和粪样进行检测，成功检出Hp DNA，靶基因16S rRNA、UreA和glmM序列与NCBI上所发表的J166和ATCC43504菌株序列的同源性均达98%以上。【结论】成功建立了特异、灵敏的牛源Hp的PCR检测方法，可以应用于临床奶牛乳样和粪样中Hp的检测。 |
| 关键词: 奶牛 幽门螺杆菌 PCR检测 16S rRNA基因 尿素酶A基因 磷酸葡萄糖胺变位酶基因 |
| DOI： |
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| 基金项目:四川省科技厅科技支撑计划项目（2011NZ0060，2013NZ0032）；四川农业大学学科建设双支计划项目（3570806） |
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| Establishment and preliminary application of a PCR method for detection of Helicobacter pylori in milk and feces of dairy cows |
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ZHANG Yu,WANG Han,WEN Yueling,et al
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| Abstract: |
| 【Objective】This research aimed to establish a specific and sensitive PCR method for the detection of Helicobacter pylori (Hp),which would provide support for the epidemiological investigation of livestock infected by Hp.【Method】A PCR method for detecting Hp contamination in clinical samples was established and optimized by designing specific primers corresponding to 16S rRNA,UreA and glmM genes of Hp strain 1 (SS1).Then,the optimized PCR method was used to detect Hp contamination in milk and feces of dairy cows.【Result】The specific test of PCR indicated that target bands could be specifically amplified from Hp SS1 by all the three pairs of primers,but unable to be amplified from Staphylococcus aureus,Salmonella,Listeria monocytogenes,Eschenchia coli and Bacillus cereus isolates.The sensitivities of PCR for amplifying 16S rRNA,UreA and glmM genes from dairy cow’s milk artificially contaminated with Hp were 101，103，and 103 CFU/mL,and they were 101，104，and 104 CFU/mL for feces samples.There were different degrees of Hp infection in the milk and feces of 41 dairy cows from large scale farms and free range farmers,and the target genes 16S rRNA,UreA and glmM had >98% homology with the published gene sequences of J166 and ATCC43504 in the GenBank database at the National Center of Biotechnology Information (NCBI). 【Conclusion】The established PCR detecting assay was specific and sensitive for Hp detection in cows,which can be applied to detect Hp contamination in clinical samples. |
| Key words: dairy cow Helicobacter pylori PCR test 16S rRNA gene UreA gene glmM gene |
