Novel method for preparation of the template DNA and selection of primers to differentiate the material rice cultivars of rice wine by PCR
Novel method for preparation of the template DNA and selection of primers to differentiate the material rice cultivars of rice wine by PCR
复制标题
DOI：
10.1016/j.jbbm.2007.07.001
复制
发表时间：
2008-04-24
影响因子：
--
通讯作者：
Nakamura, Sumiko
中科院分区：
文献类型：
--
作者：
Ohtsubo, Ken'ichi;Suzuki, Keitaro;Nakamura, Sumiko
由于许多米酒酿造商都标注了原料大米的品种名称，因此需要进行认证技术。问题是（1）发酵过程中DNA的分解，（2）微生物DNA的污染，（3）PCR抑制剂如多酚的共存。本文作者对PCR方法进行了改进：（1）将米酒冻干粉碎以浓缩DNA;（2）使用耐热淀粉酶和蛋白酶K分解淀粉和蛋白质以不影响DNA的提取;（3）用CTAB法和70%EtOH分级沉淀相结合的方法纯化模板DNA。为防止PCR扩增过程中微生物DNA的扩增，筛选出合适的植物特异性引物。从米酒中制备用于PCR的模板DNA成为可能。PCR扩增产物经测序证实与材料水稻的DNA序列完全一致。以市售黄酒为材料，采用PCR方法检测原料品种的错误标记。可以从米酒中提取和纯化用于PCR的模板DNA，并且可以使用米酒作为样品通过PCR来区分材料水稻品种。(c)2007 Elsevier B. V.保留所有权利。
As many rice wine brewers label the name of the cultivar of the material rice, authentication technology is necessary. The problems are (1) decomposition of DNAs during the fermentation, (2) contamination of DNAs from microorganisms, (3) co-existence of PCR inhibitors, such as polyphenols. The present authors improved the PCR method by (1) lyophilizing and pulverizing the rice wine to concentrate DNAs, (2) decomposition of starches and proteins so as not to inhibit DNA extraction by the use of heat-resistant amylase and proteinase K, (3) purification of the template DNA by the combination of CTAB method and fractional precipitation by 70% EtOH. To prevent the amplification of microorganism's DNAs during PCR, the present authors selected the suitable plant-specific primers. It became possible to prepare the template DNAs for PCR from the rice wine. The sequences of the amplified DNAs by PCR were ascertained to be same with those of material rice. Mislabeling of material rice cultivar was detected by PCR using the commercial rice wine. It became possible to extract and purify the template DNAs for PCR from the rice wine and to differentiate the material rice cultivars by the PCR using the rice wine as a sample. (c) 2007 Elsevier B.V. All rights reserved.