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| 发酵床垫料中微生物16S rDNA PCR反应条件的建立与优化 |
| Establishment and Optimization of Microbial 16S rDNA PCR Reaction Conditions in Fermentation Bed Padding Material |
| Received:April 24, 2014 |
| DOI：10.13254/j.jare.2014.0102 |
| 中文关键词: 发酵床垫料;16SrDNA;单因素法;退火温度 |
| 英文关键词: fermentation bed padding material;16S rDNA;single factor experiment;annealing temperature |
| 基金项目:湖南省农业产业技术体系“湖南省生猪产业技术体系生猪产业规模养殖与环境控制岗位” |
| Author Name | Affiliation | E-mail | | DU Dong-xia | Hunan Academy of Microbiology,Changsha 410009,China | | | YIN Hong-mei | Hunan Academy of Microbiology,Changsha 410009,China | | | ZHANG De-yuan | Hunan Academy of Microbiology,Changsha 410009,China | | | LIU Biao | Hunan Academy of Microbiology,Changsha 410009,China | | | XU Jun | Hunan Academy of Microbiology,Changsha 410009,China | | | WANG Zhen | Hunan Academy of Microbiology,Changsha 410009,China | | | HE Yue-lin | Hunan Academy of Microbiology,Changsha 410009,China | yyqhmm@163.com |
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| 中文摘要: |
| 为探讨发酵床垫料中微生物16S rDNA基因扩增实验中诸多因素对实验结果的影响,采用单因素法对16S rDNA基因扩增时PCR反应体系中的5个潜在因素(Mg2+、dNTP、引物、Taq酶、模板DNA)5水平上进行优化实验,并进一步优化了反应程序中的退火温度、循环次数。结果表明:25μL的最佳反应体系为:10×PCR buffer 2.5μL、MgCl22.0 mmol·L-1、dNTP 0.2 mmol·L-1、引物0.2μmol·L-1、Taq DNA聚合酶0.5 U、模板DNA 50 ng;最佳反应程序为:在94℃下进行4 min预变性;随后循环扩增25个循环(包括94℃变性45 s,53.7℃退火30 s,72℃延伸90 s);最后在72℃下延伸10 min。 |
| 英文摘要: |
| In order to study the influence of several potential factors on 16S rDNA PCR reaction conditions in fermentation bed padding mate- rial, five factors(Mg 2+, dNTP, primer, Taq DNA polymerase and DNA template)were optimized by single factor experiment at 5 concentration levels. Annealing temperatures and amplification cycle numbers were also optimized. The results showed that the optimum 25 μL reaction system comprised 10×PCR buffer 25 μL, MgCl 2 2.0 mmol· L -1, dNTP 0.2 mmol· L -1, primer 0.2 μmol· L -1, Taq DNA polymerase 0.5 U and DNA template 50 ng; and the optimum PCR procedure was as an initial pre-denaturing at 94 ℃ for 4 min, which preceded 25 cycles of dena-turing at 94 ℃ for 45 s, annealing at 53.7 ℃ for 30 s, and extension at 72 ℃ for 90 s, with a final extension at 72 ℃ for 10 min. |
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