| 不同品种猪肠道拟杆菌属-普雷沃氏菌属群实时荧光定量分析 |
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| 引用本文： | 张丽萍,王康宁.不同品种猪肠道拟杆菌属-普雷沃氏菌属群实时荧光定量分析[J].中国畜牧杂志,2009,45(7). |
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| 作者姓名： | 张丽萍 王康宁 |
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| 作者单位： | 四川农业大学动物营养所,四川,雅安,625014 |
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| 基金项目： | 国家重点基础研究发展规划(973计划) |
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| 摘 要： | 对猪肠道30种细菌的16SrDNA序列进行比对分析,设计拟杆菌属-普雷沃氏菌属荧光定量的特异引物。以多形拟杆菌菌株(CDC1404-A)基因组的DNA为模板,以常规PCR引物扩增16SrDNA靶片断,将PCR产物纯化后作为DNA标准品,将梯度稀释的标准品作模板,建立定量标准曲线。以TaqMan探针建立25μL反应体系,对不同浓度的多形拟杆菌DNA样品进行检测,并以乳酸杆菌、大肠杆菌和双歧杆菌的基因组DNA模板作阴性对照,验证此方法检测拟杆菌属-普雷沃氏菌属的特异性。结果表明:设计的荧光定量引物和探针,仅对拟杆菌和普雷沃氏菌有很强的特异性,该方法的灵敏度可3.73个/μL的基因组DNA。将PCR方法用于5月龄长白和太湖猪回肠内容物检测,其拟杆菌属-普雷沃氏菌属的检测结果呈阳性,每克回肠内容物拟杆菌属-普雷沃氏菌属数量的拷贝数对数值太湖猪比长白猪高38.5%(P<0.10),而且发现拟杆菌属-普雷沃氏菌属数量与背膘厚度、脂肪率之间有正相关关系。
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| 关 键 词： | 拟杆菌属-普雷沃氏菌属 实时荧光定量PCR(FQ-PCR) TaqMan探针 |
Establishment and Application of Fluorescence Quantitative PCR for Detecting Swine Bacteroides-prevotella Population |
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| ZHANG Li-ping,WANG Kang-ning.Establishment and Application of Fluorescence Quantitative PCR for Detecting Swine Bacteroides-prevotella Population[J].Chinese Journal of Animal Science,2009,45(7). |
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| Authors: | ZHANG Li-ping WANG Kang-ning |
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| Abstract: | Based upon multiple sequence alignment of the 16S rDNAs of 30 bacteria in pig tract,the fluorescence quantitative specific primers and probe were designed for deceting bacteroides-prevotella.The DNA of B.thetaiotaomicron(CDC1404-A)was used as template,the target 16S rDNA fragment was amplified by PCR with specific primers,and used as standard sample,the dilution gradients of which were subjected to the standard quantitation curve in FQ-PCR assay.Different concentrations of B.thetaiotaomicron genomic DNA were detected by FQ-PCR in a 25 μL reaction system with TaqMan probe.At the same time,various genomic DNAs of lactobacillus, escherichia, bifidobacterium were used as negative controls to confirm specificity of the FQ-PCR detection assay.Results demonstrated that the designed primers and probe were of high homology only with bacteroide and prevotella 16S rDNA, and that sensitivity of the FQ-PCR assay was of 3.73cells/μL of bacterial DNA.The quantity of bacteroides in the ileum of Landrace and Taihu pig at 5 month was detected.Bacteroides-prevotella was positive in all illeum content samples.The logarithm for the numbers of bacteroides-prevotella per gram illeum content in Taihu pig was higher 35%(P < 0.10) than that of Landrace,and the positive correlation exsited in the number of bacteroides-prevotella with backfat thickness and fat percentage. |
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| Keywords: | Bacteroides-prevotella fluorescence quantitative PCR TaqMan probe |
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