JP4957164B2 - Method for determining the ratio of different varieties in rice - Google Patents

Description

  The present invention relates to a technique for discriminating rice varieties based on rice grain deoxyribonucleic acid (hereinafter referred to as “DNA”), and more particularly to a method for calculating the proportion of different varieties of rice grains mixed in a predetermined variety of rice grains. It is.

  Conventionally, for example, Patent Document 1 by the inventor of the present application is known as a technique for determining a variety of rice grains by extracting DNA from the rice grains. The cultivar determination method according to Patent Document 1 is to determine the varieties for one rice grain. The method of determining the variety of Patent Document 1 pulverizes one grain of rice to be examined, extracts DNA from the pulverized rice grain by a known CTAB (cetyl-trimethyl-ammonium bromide) method (hereinafter referred to as “CTAB method”), Thereafter, a paired pairing primer that binds only to the base sequence site serving as an identification band useful for identifying the variety is added to the extracted DNA (template DNA), and PCR (polymerase chain reaction) is added. (Hereinafter referred to as “PCR method”) to obtain a PCR product in which the nucleotide sequence site is amplified. Thereafter, the PCR product is subjected to electrophoresis (agarose electrophoresis) to detect a product-specific identification band and to perform product determination.

JP 2005-73655 A

As described above, the method of scientifically determining the variety of rice grains is useful for analyzing whether or not the display of the variety of polished rice circulated in the market is illegal, and has attracted attention recently. However, since the above-mentioned variety determination method is for determining the variety of rice grains (inspected rice grains) in units of one grain, for example, a plurality of assumed variety (varieties displayed on a polished bag of polished rice) When investigating whether or not rice grains of different varieties other than the above-mentioned varieties are mixed in the rice grain group and the proportion of rice grains of the mixed different varieties, the work time for the investigation and measurement is long. There was a problem of time.
Accordingly, in view of the above problems, the present invention provides a method for calculating the mixing ratio of different varieties in rice grains, which can determine in a short time the proportion of rice grains of different varieties mixed in a plurality of rice grains of assumed varieties. This is a technical issue.

In order to solve the above problem, the invention of claim 1
Virtual grinding a plurality of rice grain sample rice grains is mixed in different varieties of rice grains of constant varieties, the DNA extraction step of extracting DNA from該米grains grind
Designed to detect a common peak for a plurality of varieties in the DNA extracted by the DNA extraction step, a positive primer comprising a pair of SEQ ID NO: 13 and SEQ ID NO: 14 in the sequence listing and rice grain varieties specific SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: A DNA amplification step in which a PCR method is performed by adding a plurality of pairing primers each consisting of No. 10, SEQ ID No. 11 and SEQ ID No. 12 as a pair;
Performed microchip electrophoresis based on PCR products amplified by the DNA amplification step, except for the peak indicated by the assumed varieties among the peaks detected by the microchip electrophoresis peaks of the different varieties The different product peak detection process to be identified,
A peak intensity value detecting step for obtaining a peak intensity value of a different variety peak identified by the different variety peak detecting step and a peak intensity value of a positive control detected by addition of the positive primer,
A division step of dividing the different product peak intensity value detected by the peak intensity value detection step by the peak intensity value of the positive control;
Based on a calibration curve indicating the relationship between the division value and the mixture ratio of different varieties obtained in advance for each of the paired primers, the mixture of different varieties for determining the mixture ratio of different varieties in the division value calculated in the division step A ratio determination step;
Taken technical measures including.

According to the method of claim 1,
DNA is extracted from a pulverized rice grain obtained by pulverizing a plurality of rice grain samples to be examined, and the extracted DNA is amplified and subjected to microchip electrophoresis. Each peak and peak intensity value of the variety is specified. When amplifying the DNA, a positive primer designed to detect a peak at a common position (base size bp) for a plurality of rice varieties is added. The present invention has found the knowledge that there is a relative proportional relationship between the peak intensity value of the peak detected by the addition of the positive primer (positive control = positive control) and the peak intensity value of different varieties. Is used to determine the mixture ratio of different varieties. Therefore, when determining the different product mixture ratio, the division value, which is the ratio between the positive intensity peak intensity value and the different product peak intensity value, was calculated, and the relationship between the division value and the different product mixture ratio was expressed. A calibration curve is obtained in advance, and based on the calibration curve, a value corresponding to the calculated division value (different product mixture ratio) is determined.

The front Symbol positive primers took technical means to primers and SEQ ID NO: 13 and SEQ ID NO: 14 in Sequence Listing in a pair. The discrimination of the type is within a predetermined range of the DNA amplification size (bp), which is the horizontal axis of the output of the microchip electrophoresis, among the peaks obtained by performing the microchip electrophoresis. According to the positive primer according to claim 2, the positive control peak is detected at a lower limit value (for example, 100 bp) of the predetermined range. The base sequence is designed. For this reason, by adjusting the concentration and amount of positive primer to increase the peak intensity of the positive control, the distinction from the non-specific product peak detected as noise near the lower limit becomes clear. The lower limit value (lower marker) becomes clear.

Furthermore, before Kitaigo primer sequence in sequence listing, SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and SEQ ID NO: 5 SEQ ID NO: 6, and SEQ ID NO: 7 SEQ ID NO: 8, and SEQ ID NO: 9 The technical means of using the primer which makes a pair each number 10, sequence number 11, and sequence number 12 was taken. Thereby, by using the paired primer, a suitable action for obtaining the effect of the present invention can be obtained in the DNA amplification step.

  According to the method of the present invention, different rice varieties are mixed at a time based on a plurality of rice grains, rather than determining (calculating) the mixing ratio of different varieties by determining the variety of rice grains to be inspected one by one. Since the ratio can be determined, the working time required for the determination can be shortened.

  The assumed varieties in the present invention indicate the varieties displayed on the polished rice packaging bags and the like distributed in the market. Hereinafter, an embodiment of the present invention will be described with reference to the flowchart of the method for calculating the mixture ratio of different varieties of the present invention shown in FIG.

Rice grain sample preparation:
50 grams (several tens of grams) of about 9% of “Koshihikari” (assumed variety) mixed with “Kirara 397, which is a different variety other than the assumed variety”, as a rice grain sample for inspection (raw material) of the example ) Prepared, pulverized, and 0.1 gram (small amount) taken out from the pulverized product was used as a measurement sample (hereinafter referred to as “rice pulverized product”).

DNA extraction step (S1):
A DNA extraction step is performed based on the pulverized rice grains. The DNA extraction step is performed by the commonly used CTAB method. In the CTAB method, beads are placed in a predetermined container (tube), crushed using a crushing device (Kurashita Boseki Co., Ltd .: sample crusher <SH-100>), and further, a plant cell lysis reagent (main component: Tris, EDTA, NaCl, CTAB) are added and heated for 1 hour to dissolve the rice plant cells. Thereafter, the container is set in an automatic DNA separation device (manufactured by Kurashiki Boseki Co., Ltd .: nucleic acid automatic separation device, model: PI-24). A few milliliters of plant protein denaturing reagent (main component: SDS, other surfactants) is added and stirred to denature the rice grain protein, followed by plant protein denaturing reagent (main component: chloroform (90%)) ) Is added and stirred to elute the lipid of rice grains. Next, after adding and stirring a few mL of plant deproteinization reagent (main component: potassium acetate), a centrifugal action is applied to form a pellet containing chloroform, plant residues, etc., and the transfer of the supernatant is made smooth .

  Next, the supernatant is transferred to a collection container containing several mL of nucleic acid complex precipitation reagent (main components: Tris, EDTA, CTAB) in advance and stirred to form a CTAB-DNA complex. Then, after giving a centrifugal action, the supernatant is discarded. Next, several mL of a nucleic acid redissolving reagent (main component: NaCl) is added and stirred to dissociate the CTAB-DNA complex. Next, several mL of a precipitation reagent (main component: isopropanol (90% or more)) is sequentially added, stirred and centrifuged to precipitate DNA. Thereafter, the supernatant is discarded again. Further, several mL of a washing reagent (main component: ethanol (70% or less), isopropanol) is added, and the DNA is washed by stirring and centrifuging. Then, again, the supernatant is discarded and finally dried by centrifugation to obtain template DNA.

DNA amplification step (S2):
Next, a DNA amplification step is performed based on the template DNA. The DNA amplification step is also performed by the generally used PCR method. In performing the PCR method, first, several tens of μL of a preparation solution having the composition shown in Table 1 below is prepared in a predetermined container.
(Table 1)
Sterile water 10 × PCR buffer (100 mM Tris HCL (pH 8.3), 500 mM KCL)
MgCl2
dNTP mix (2.5 mM each)
First paired primer (SEQ ID NO: 1) forward side First paired primer (SEQ ID NO: 2) reverse side Second paired primer (SEQ ID NO: 3) forward side Second paired primer (SEQ ID NO: 4) reverse side Third Paired primer (SEQ ID NO: 5) Forward side Third paired primer (SEQ ID NO: 6) Reverse side Fourth paired primer (SEQ ID NO: 7) Forward side Fourth paired primer (SEQ ID NO: 8) Reverse side 5th paired Primer (SEQ ID NO: 9) Forward side Fifth paired primer (SEQ ID NO: 10) Reverse side Sixth paired primer (SEQ ID NO: 11) Forward side Sixth paired primer (SEQ ID NO: 12) Reverse side Positive primer (SEQ ID NO: 13) ) Forward side Positive primer (SEQ ID NO: 14) Reverse side Template DNA solution Taq polymerase (5 U / μL

The base sequence of each paired primer (SEQ ID NO :) is as follows.
(Chemical formula 1)
gaacaattac tccctcggtt ctata (SEQ ID NO: 1)
(Chemical formula 2)
gcatgagcgg catgacagaa (SEQ ID NO: 2)
(Chemical formula 3)
cactgtaact tttgtaagtt acactg (SEQ ID NO: 3)
(Chemical formula 4)
tcccccgttc aatgagaagc t (SEQ ID NO: 4)
(Chemical formula 5)
cctcttgtat cctgtccccc tc (SEQ ID NO: 5)
(Chemical formula 6)
agttggcacg tggtgcagaa (SEQ ID NO: 6)
(Chemical formula 7)
gcatccatcc tggcctagcg ctgtata (SEQ ID NO: 7)
(Chemical Formula 8)
gcaggtcgaa aacacacaga acgatac (SEQ ID NO: 8)
(Chemical 9)
cgctccatgc acacatgagc tag (SEQ ID NO: 9)
(Chemical formula 10)
cctatgctgt gatcttgtag tgc (SEQ ID NO: 10)
(Chemical 11)
catgccacat cgtcagagca ctcg (SEQ ID NO: 11)
(Chemical formula 12)
gtgcaatcca ttgctgaata ag (SEQ ID NO: 12)
(Chemical 13)
ggactaccaa ccccagctac (SEQ ID NO: 13)
(Formula 14)
ccagctgggt atcctaggag a (SEQ ID NO: 14)

As described above, a positive primer is used in the composition of the preparation solution. The positive primer has a base sequence designed so that a common peak is detected in a plurality of varieties when analyzed by a microchip electrophoresis system described later. In the positive primer of this example, the base sequence is designed so that a peak (positive control) is detected at, for example, 100 base size (bp) which is the lower limit (lower marker) of the peak detection range, and 100 In order to clarify the distinction from the peaks of different varieties of rice grains (non-specific products) detected as many noises near the base size, the peak intensity value is increased by adjusting the concentration and addition amount. The six types (first to sixth) of the paired primers are designed so that, if there is a reaction, a peak appears at each different base size (horizontal axis in FIG. 2 ).

  In addition, in the above preparation solution, in order to ensure reproducibility, the concentration and the addition amount of the positive primer and the pairing primer to be added are always set to predetermined amounts, and the magnitude of each detected peak intensity value is relative. However, it is necessary to prevent the intensity values of adjacent peaks from reversing even if they go up and down.

  Next, a container containing a preparation liquid having the composition shown in Table 1 was set in a PCR device (ABI, Gene Amp PCR System 9700), heated at 95 ° C. for 2 minutes to be thermally denatured, and further heat denatured ( 95 ° C. × 2 minutes), annealing (62 ° C. × 1 minute), and extension reaction (72 ° C. × 2 minutes) are repeated 30 cycles to carry out the extension reaction (72 ° C. × 4 minutes). After the extension reaction (72 ° C. × 4 minutes), the solution is allowed to stand until it reaches 4 ° C. Thus, a PCR product is obtained.

Non-specific peak detection step (different product peak detection step) (S3):
Next, using the PCR product, a non-specific peak detection step (different variety peak detection step) S3 is performed to identify the peak shown by reaction of different varieties of rice grains. In the non-specific peak detection step S3, analysis is performed using a microchip electrophoresis system (trade name: SV1210 Cosmo Eye, manufactured by Hitachi Chemical Co., Ltd.), which is a known device. FIG. 2 shows the results of analysis by setting the PCR product in the microchip electrophoresis system. On the other hand, FIG. 3 shows the peak pattern and positive control indicated by “Koshihikari” (assumed variety) when using the above-mentioned six kinds of paired primers and positive primer as in this example. It is shown. From FIG. 3, it can be seen that “Koshihikari” has a peak at a position of 548 base size and shows a reaction only to the second pairing primer. In addition, for the five types of pairing primers below the second pairing primer, if there was a reaction,
The first pairing primer is at the position of 1046 base size,
The third pairing primer is at the position of 1596 base size,
The fourth pairing primer is at a position of 409 base size,
The fifth pairing primer is at a position of 626 base size,
The sixth pairing primer is at a position of 1354 base size,
The base sequence is designed so that each peak is detected.

  The non-specific peak detection step S3 is based on FIG. 2 and FIG. 3, and from the plurality of peaks shown in FIG. 2, the Koshihikari peak of the assumed variety shown in FIG. First, a peak appearing at 548 bases (specific peak) is removed. As a result of the removal, in this example, three different variety peaks (hereinafter referred to as “non-specific peaks”) indicated by different varieties of rice grains are detected, and thus the rice grain sample contains different varieties of rice grains. Is shown. The first of the different types of peaks is a peak of 1046 base size (hereinafter referred to as “non-specific peak A”), which is a peak showing a reaction with the first paired primer. The second is a peak of 1354 base size (hereinafter referred to as “non-specific peak B”), which is a peak showing a reaction with the sixth paired primer. The third is a peak with a size of 1596 bases (hereinafter referred to as “non-specific peak C”), which is a peak showing a reaction with the third pairing primer (FIG. 2). Note that the peak L detected in FIGS. 2 and 3 is an upper marker and is not a specific peak or a non-specific peak. Similarly, the positive control (hereinafter referred to as “positive control”) detected by adding the positive primer detected in FIGS. 2 and 3 is not a specific peak or a non-specific peak.

Peak intensity value detection step (S4):
Next, in the peak intensity value detecting step, the peak intensity values of the non-specific peaks A, B, C and positive control shown in FIG. 2 are detected. The detection results are as follows.
Each peak intensity value:
Non-specific peak A: 503
Non-specific peak B: 414
Non-specific peak C: 378
Positive control: 3959

Division step (S5):
Next, as the division step, the peak intensity values of the non-specific peaks A, B, and C are divided by the peak intensity value of the positive control based on the peak intensity values detected in the peak intensity value detection step. The result of this division is as follows:
Division result:
(1) Division value of non-specific peak A Non-specific peak A / positive control = 503/3959 = 0.127
(2) Divided value of non-specific peak B Non-specific peak B / positive control = 414/3959 = 0.105
(3) Division value of non-specific peak C Non-specific peak C / positive control = 378/3959 = 0.095

Different kind mixture ratio determination step (S6):
Next, the different kind mixing ratio determining step determines the mixing ratio of different kinds using the division value and a calibration curve (described later). The calibration curve is obtained in advance for each of the first to sixth paired primers. In preparing the calibration curve, first, the preparation solution (the DNA amplification step (S2)) in which the positive primer is mixed with a single paired primer is prepared, and a variety 1 in which no peak is detected in the preparation solution. And the product type 2 from which the peak is detected are obtained in advance. Each rice grain sample is prepared by mixing the cultivar 1 (the peak is not detected) with the cultivar 2 (the peak is detected) at a ratio of 10%, 20%, 30%, etc., respectively. The division value is calculated from the peak intensity value of the varieties 2 and the positive intensity of the positive control, and a calibration curve is created from the relationship between the rice grain samples having different mixing ratios of the varieties 2 and the division values. In the same manner, a calibration curve is obtained in advance for other single paired primers. Figure 4 (1) shows a calibration curve and its contribution R ² of the first paired primers that detected the non-specific peaks A. (1) in FIG. 5 shows a calibration curve of the sixth mating primer detecting the nonspecific peaks B and its contribution ratio R ^2. Further, in FIG. 6 (1) shows a calibration curve of the third mating primers detect nonspecific peak C and its contribution ratio R ^2. When the mixing ratio of different varieties is determined based on the calibration curves of the first, sixth, and third paired primers and the division values of the non-specific peaks A, B, and C, the following results are obtained. It becomes.
(1) According to the calibration curve of the first paired primer (FIG. 4), the heterogeneous mixture ratio with respect to the division value 0.127 of the non-specific peak A is 9%.
(2) According to the calibration curve of the sixth paired primer (FIG. 5), the mixture ratio of different varieties with respect to the division value 0.105 of the non-specific peak B is 8%.
(3) According to the calibration curve of the third paired primer (FIG. 6), the mixture ratio of different varieties with respect to the division value 0.059 of the non-specific peak C is 8%.

  From the above, it is determined that the heterogeneous mixture ratio in the nonspecific peak A is 9%, the heterogeneous mixture ratio in the nonspecific peak B is 8%, and the heterogeneous mixture ratio in the nonspecific peak C is 8%. For this reason, the mixing rate of different varieties in the rice grain sample (the rice grain group to be inspected) of this example is determined as 8% to 9%, which is within the range from the lowest value to the highest value. This determination result coincides with “Koshihikari” (assumed variety), which is a rice grain sample of this example, mixed with about 9% “Kirara 397, which is a different variety other than the assumed variety”.

  In addition, the determination of the mixture ratio of different varieties in the present invention is not limited to the range from the highest value to the lowest value as described above, but may be an average value of the highest value and the lowest value. Furthermore, in this example, three non-specific peaks were detected, but depending on the rice grain sample, there may be other than three. Thus, even if the number of detected non-specific peaks is other than three, there is no problem, and it is sufficient to determine the mixing ratio of different varieties by the same process as described above.

The flowchart explaining the different kind mixing ratio determination method in the rice grain of this invention. In the Example, the result of having applied to the microchip electrophoresis the rice grain sample which added "other different varieties (Kirara 397)" to "Koshihikari" (expected varieties). In Example, “Koshihikari” was subjected to microchip electrophoresis. (1) is a calibration curve for the first paired primer. (2) is an example in which the mixing rate of different varieties was determined using the same calibration curve. (1) is a calibration curve for the sixth paired primer. (2) is an example in which the mixing rate of different varieties was determined using the same calibration curve. (1) is a calibration curve for the third paired primer. (2) is an example in which the mixing rate of different varieties was determined using the same calibration curve.

Explanation of symbols

S1 DNA extraction step S2 DNA amplification step S3 Non-specific peak detection step (different kind peak detection step)
S4 Peak intensity value detection step S5 Division step S6 Different kind mixing ratio determination step

Claims (1)

Virtual grinding a plurality of rice grain sample rice grains is mixed in different varieties of rice grains of constant varieties, the DNA extraction step of extracting DNA from該米grains grind
Designed to detect a common peak for a plurality of varieties in the DNA extracted by the DNA extraction step, a positive primer comprising a pair of SEQ ID NO: 13 and SEQ ID NO: 14 in the sequence listing and rice grain varieties specific SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: A DNA amplification step in which a PCR method is performed by adding a plurality of pairing primers each consisting of No. 10, SEQ ID No. 11 and SEQ ID No. 12 as a pair;
Performed microchip electrophoresis based on PCR products amplified by the DNA amplification step, except for the peak indicated by the assumed varieties among the peaks detected by the microchip electrophoresis peaks of the different varieties The different product peak detection process to be identified,
A peak intensity value detecting step for obtaining a peak intensity value of a different variety peak identified by the different variety peak detecting step and a peak intensity value of a positive control detected by addition of the positive primer,
A division step of dividing the different product peak intensity value detected by the peak intensity value detection step by the peak intensity value of the positive control;
Based on a calibration curve indicating the relationship between the division value and the mixture ratio of different varieties obtained in advance for each of the paired primers, the mixture of different varieties for determining the mixture ratio of different varieties in the division value calculated in the division step A ratio determination step;
A method for calculating the mixing ratio of different varieties in rice grains.

Images