| 瘤胃总甲烷菌实时荧光定量PCR方法的构建 |
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| 引用本文： | 李长青,金海,栗原光规.瘤胃总甲烷菌实时荧光定量PCR方法的构建[J].黑龙江畜牧兽医,2009(5). |
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| 作者姓名： | 李长青 金海 栗原光规 |
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| 作者单位： | 李长青,金海,LI Chang-qing,JIN Hai(内蒙古农牧业科学院,生物中心,内蒙古,呼和浩特,010031);栗原光规,KURIHARA Mitsunori(日本国家农业生物资源研究所,动物科学研究组,日本,茨城,305-0901)
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| 摘 要： | 试验构建了瘤胃总甲烷菌实时荧光绝对定量PCR的标准品及标准曲线用于总甲烷菌的定量测定,并提取瘤胃微生物总DNA, 以甲烷菌特异性引物进行PCR扩增, 回收纯化PCR产物,与pBS-T载体连接并转化到大肠杆菌,再用氨苄西林培养基筛选阳性重组质粒, 提取含目的片段质粒DNA, 通过PCR及测序鉴定重组质粒.根据OD值确定浓度, 将梯度稀释的质粒作为模板, 进行荧光定量PCR反应并作出标准曲线.结果表明: 所构建的标准曲线具有很高的相关性(R2=0.992), 并获得了高扩增效率产物.
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| 关 键 词： | 瘤胃甲烷菌 实时荧光定量PCR 标准品质粒 标准曲线 |
Development of a real-time Absolute quantitative PCR for quantification of total methanogens in rumen |
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| LI Chang-qing,JIN Hai,KURIHARA Mitsunori.Development of a real-time Absolute quantitative PCR for quantification of total methanogens in rumen[J].Heilongjiang Animal Science And veterinary Medicine,2009(5). |
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| Authors: | LI Chang-qing JIN Hai KURIHARA Mitsunori |
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| Institution: | 1.Biotechnology Research Center;Inner Mongolia Academy of Agricultural and Animal Husbandry Science;Huhhot 010031;China;2.Division of Animal Sciences;National Institute of Agrobiological Sciences;Ibaraki 305-0901;Japan |
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| Abstract: | To quantify the population sizes of total methanogens in rumen,the standard plasmids and curves of the species was constructed using Real-time Absolute quantitative PCR.Ruminal microbial total DNA was isolated from rumen content in the Inner Mongolia goat,linked with pBS-T Vector to construct recombinant plasmid and transfected into Escherichia coli.Target plasmids in white colonies were screened by ampicillin screening,and their specificity was identified by direct PCR and DNA sequencing.Plasmid DNA was ex... |
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| Keywords: | Ruminal methanogens Real-time PCR standard plasmid standard curve |
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