Method of detecting the presence of absence of mixed varieties in grains, and identifying the mixed varieties
a technology of mixed varieties and grains, which is applied in the field of detecting the presence or absence of mixed varieties in grains, and identifying the mixed varieties, can solve the problems of difficult individual detection and identification of those different varieties of mixed rice, and the inability to detect the presence or absence of any different varieties mixed in a certain variety of rice, and achieve the effect of rapid and accurate detection of the presence and absen
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example 1 (
Detection of Different Varieties Mixed in “Koshihikari” by the Use of DNA Groups for Negative Band Discrimination)
[0111] Best twenty varieties of rice harvested in 1999 in Japan, “Koshihikari”, “Hitomebore”, “Hinohikari”, “Akitakomachi”, “Kirara 397”, “Kinuhikari”, “Hoshinoyume”, “Haenuki”, “Mutsuhomare”, “Nipponbare”, “Sasanishiki”, “Tsugaruroman”, “Hanaechizen”, “Yumetsukushi”, “Hatsushimo”, “Asanohikari”, “Tsukinohikari”, “Aichinokaori”, “Matsuribare” and “Akiho” were tested, all unpolished. Using a laboratory rice mill, (Yamamoto Seisakusho's Ricepal VP31T), these were milled into polished rice to a yield of 90%.
[0112] Using an ultracentrifugal grinder (by Retsch), the polished rice samples were separately ground. A genome DNA was extracted from 6 g of each powder sample through CTAB treatment. Concretely, 6 ml of a 2% CTAB solution (0.1 M tris-HCl, 2 mM disodium ethylenediaminetetraacetate (EDTA), 1.4 M NaCl, pH 8.0) at 70° C. was added to the sample and stirred, and put into ...
example 2 (
Discrimination of Different Varieties in Mixed Rice by the Use of DNA Groups for Negative / Positive Band Discrimination)
[0118] Best twenty varieties of rice harvested in 1999 in Japan, “Koshihikari”, “Hitomebore”, “Hinohikari”, “Akitakomachi”, “Kirara 397”, “Kinuhikari”, “Hoshinoyume”, “Haenuki”, “Mutsuhomare”, “Nipponbare”, “Sasanishiki”, “Tsugaruroman”, “Hanaechizen”, “Yumetsukushi”, “Hatsushimo”, “Asanohikari”, “Tsukinohikari”, “Aichinokaori”, “Matsuribare” and “Akiho” were tested, all unpolished. Using a laboratory rice mill, (Yamamoto Seisakusho's Ricepal VP31T), these were milled into polished rice to a yield of 90%. One grain of each polished rice sample was ground in a mortar to prepare powder rice samples. A genome DNA was extracted from each powder sample, using a DNA extraction kit, ISOPLANT II.
[0119] A PCR composition was prepared by mixing 10.4 μl of sterilized water, 0.2 μl of polymerase (Taq polymerase (5 U / μl) by Takara Bio Inc.), 2.5 μl of PCR buffer (12 mM tris-HCl...
example 3 (
Detection of Mixed Rice by the Use of Three DNA for Negative Band Discrimination)
[0124] Best twenty varieties of rice harvested in 1999 in Japan, “Koshihikari”, “Hitomebore”, “Hinohikari”, “Akitakomachi”, “Kirara 397”, “Kinuhikari”, “Hoshinoyume”, “Haenuki”, “Mutsuhomare”, “Nipponbare”, “Sasanishiki”, “Tsugaruroman”, “Hanaechizen”, “Yumetsukushi”, “Hatsushimo”, “Asanohikari”, “Tsukinohikari”, “Aichinokaori”, “Matsuribare” and “Akiho” were tested, all unpolished. In the same manner as in Example 1, a template DNA was prepared from each polished rice sample.
[0125] In this Example, mixed rice of “Koshihikari” with a small amount of a different variety was estimated and tried. For this, 80% of DNA of “Koshihikari” was mixed with 20% of DNA of any other different variety to prepare a template DNA. The Template DNA was subjected to PCR with a primer mixture of S13F25 and S13R24 (SEQ ID Nos. 83 and 84) of 0.08 μl each; E30F28 and E30R24 (SEQ ID Nos. 39 and 40) of 0.05 μl each; and M2CGF16...
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