Novel method for preparation of the template DNA and selection of primers to differentiate the material rice cultivars of rice wine by PCR

Abstract

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.