CN111118209A - Dual real-time fluorescence PCR identification method for indica-japonica subspecies rice based on chloroplast DNA difference - Google Patents
Dual real-time fluorescence PCR identification method for indica-japonica subspecies rice based on chloroplast DNA difference Download PDFInfo
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Abstract
The invention provides a double real-time fluorescence PCR (polymerase chain reaction) rapid identification method of indica-japonica subspecies rice varieties based on chloroplast DNA sequence difference. On the basis of a pair of published primers, 1 probe 5 '-HEX-ATG CAA TAG AGA GCG AGT GG-BHQ 1-3' is designed, a double real-time fluorescence PCR identification method of indica-japonica subspecies rice is established by using the published primers and probes for real-time fluorescence PCR detection of endogenous gos9 genes of rice in a standard, only 1 fluorescence amplification curve of the endogenous gos9 gene of rice appears in the PCR amplification result of cpDNA-deleted indica rice, 2 fluorescence amplification curves of the endogenous gos9 gene of rice and japonica rice-specific cpDNA simultaneously appear in the PCR amplification result of cpDNA-non-deleted japonica rice, and the curve difference is used for effectively identifying the type of the rice to be detected.
Description
Technical Field
The invention relates to a double real-time fluorescence PCR (polymerase chain reaction) rapid identification method of indica-japonica subspecies rice varieties based on chloroplast DNA (chloroplast DNA, cpDNA) difference.
Background
Rice is staple food in most countries, also in southern China, and is also the main raw material of important processed food, and mainly comprises 2 subspecies, namely indica (rice) subspecies ((rice))Hsienrice, also known internationally asIndicarice and round-grained nonglutinous rice subspecies (rice) ()Kengrice, also known internationally asJaponicarice) of different varieties and different commodity values, it is necessary to perform discrimination. From the morphology of the rice, 2 subspecies have certain differences, but the precise identification is difficult after the rice is processed into the rice, and the rice product processed after the rice is crushed can not be identified morphologically at all. Therefore, it is necessary to establish a DNA identification method of rice species. The invention relates to a duplex fluorescence PCR (polymerase chain reaction) rapid detection method for varieties of indica-japonica subspecies rice based on cpDNA difference.
The establishment of the method has important significance for identifying the rice varieties of the rice seeds, the rice and the processed products thereof, maintaining the food market order at home and abroad and protecting the benefits of the rice seeds, the rice and the processing enterprises and consumers.
Disclosure of Invention
The invention aims to provide a dual real-time fluorescence PCR rapid identification method of indica-japonica subspecies rice varieties based on cpDNA difference.
The invention comprises the following contents:
1. based on the prior invention, on the basis of self-designing primers for identifying indica rice and japonica rice types, 1 probe is designed on a specific 69bp DNA fragment of indica rice deletion and japonica rice, and is used for establishing a double real-time fluorescence PCR identification method of indica rice and japonica rice, and the sequence is as follows: 5 '-HEX-ATG CAA TAG AGA GCG AGT GG-BHQ 1-3';
2. optimizing a double real-time fluorescent PCR reaction system, and establishing a double real-time fluorescent PCR identification method for identifying indica rice and japonica rice. Reaction system: 2 XMix 10. mu.L, primer gos 9-F0.2. mu. L, gos 9-R0.2. mu.L, probe gos 9-P0.1. mu.L, primer rice-F40.6. mu.L, rice-R10.6. mu.L, probe rice-P0.3. mu.L, DNA (100 ng/. mu.L) 2. mu.L, ddH2O6.0. mu.L. Reaction conditions are as follows:35s at 95 ℃; 10s at 95 ℃, 40s at 60 ℃ and 40 cycles.
The primer gos9-F is 5'-TTA GCC TCC CGC TGC AGA-3', gos9-R is 5'-AGA GTC CACAAG TGC TCC CG-3', and the probe gos9-P is 5 '-FAM-CGG CAG TGT GGT TGG TTT CTT CGG-TAMRA-3'; rice-F4 is 5'-AAT CGC AAC CCC TTT CCG C-3', rice-R1 is 5'-TTG AGG ATT ATT CCATGA TTC C-3', and probe rice-P is 5 '-HEX-ATG CAA TAG AGA GCG AGT GG-BHQ 1-3'.
Only 1 amplification curve of the rice species specific endogenous gos9 gene appears in the double real-time fluorescence PCR result of the indica rice, 2 amplification curves of the rice endogenous gos9 gene and the japonica rice specific cpDNA appear in the double real-time fluorescence PCR result of the japonica rice, and the difference of the amplification curves is used for judging the subspecies to which the rice to be detected belongs.
The invention has the advantages that:
the invention provides a double-fluorescence PCR rapid detection and identification method of indica-japonica rice varieties based on cpDNA sequence difference. The difference of the sequence of the cpDNA of the indica-japonica rice is utilized, namely, a specific probe is designed at the position of a 69bp segment (non-deleted) of chloroplast DNA of the indica-japonica rice, forward and reverse primers (applied patent) are designed at two ends of the difference segment, and a primer and probe combination for synthesizing and amplifying endogenous gos9 genes of rice is synthesized in an SN/T2584-. The establishment of the method has important significance for identifying the types of rice subspecies of rice seeds, rice and processed products thereof, maintaining the food market order at home and abroad and protecting the benefits of the rice seeds, the rice and the processed enterprises and consumers.
Drawings
FIG. 1 shows the positions of primers and probes for indica-japonica rice identification.
FIG. 2 shows a double real-time fluorescence PCR curve diagram of indica rice (a) and japonica rice (b) with amplification primers and probes of endogenous gos9 gene of rice and japonica rice specific cpDNA fragment in a ratio of 1: 1.
FIG. 3 shows a double real-time fluorescence PCR curve diagram of indica rice (a) and japonica rice (b) with amplification primers and probes of endogenous gos9 gene of rice and japonica rice specific cpDNA fragment in a ratio of 1: 2.
FIG. 4 shows a double real-time fluorescence PCR curve diagram of indica rice (a) and japonica rice (b) with amplification primers and probes of endogenous gos9 gene of rice and japonica rice specific cpDNA fragment in a ratio of 1: 3.
FIG. 5 shows a double real-time fluorescence PCR curve diagram of indica rice (a) and japonica rice (b) with amplification primers and probes of endogenous gos9 gene of rice and japonica rice specific cpDNA fragment in a ratio of 1: 4.
FIG. 6 shows a double real-time fluorescence PCR curve diagram of indica rice (a) and japonica rice (b) with amplification primers and probes of endogenous gos9 gene of rice and japonica rice specific cpDNA fragment in a ratio of 1: 5.
FIG. 7 shows the results of duplex real-time fluorescence PCR of the endogenous gos9 gene of indica rice and the cpDNA fragment specific to japonica rice, and only 1 amplification curve of the endogenous gos9 gene of indica rice (about 45 samples) appears.
FIG. 8 shows the results of double real-time fluorescence PCR of the endogenous gos9 gene of japonica rice and the cpDNA fragment specific to japonica rice, wherein japonica rice (about 30 samples) shows 2 amplification curves of the endogenous gos9 gene of japonica rice and the cpDNA fragment specific to japonica rice.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the following examples are only examples of the present invention and do not represent the scope of the present invention defined by the claims.
Example 1
1 test Material
Rice seeds were collected by this laboratory.
2 Primary reagent
CTAB lysate [ weighing 4.00 g CTAB, 16.38 g NaCl, 2.42 g Tris, 1.50 g Na2EDTA, dissolving with appropriate amount of water, adjusting pH to 8.0, diluting to 200mL, and autoclaving]CTAB precipitation solution (1.00 g CTAB and 0.50 g NaCl are weighed, dissolved by a proper amount of water, the pH value is adjusted to 8.0, the volume is adjusted to 200mL,autoclaving]Self-preparation; premix Ex TaqTM(Probe qPCR) is a product of TaKaRa, Dalian bioengineering (Dalian) Co., Ltd.
3 main apparatus and equipment
A grinder, a vortex apparatus, a water bath, a bench top centrifuge, a large refrigerated centrifuge, a real-time fluorescent quantitative PCR apparatus (BIO-RAD, Inc., CFX96, USA).
4 primer and probe
Primers and probes for amplifying endogenous gos9 genes of rice are synthesized according to the standard SN/T2584-.
According to the previous research, on the basis of self-designing and identifying the primer (rice-F4 and rice-R1) of indica rice and japonica rice varieties, a sequence which accords with the design principle of a TaqMan probe is selected from a 69bp DNA fragment which is peculiar to indica rice and japonica rice and is used as a probe (rice-P) for amplifying a japonica rice specific cpDNA fragment. The probe is not continuously matched with 4 bases of a primer and a probe of a gos9 gene to identify indica rice and japonica rice types (rice-F4 and rice-R1), and a secondary structure cannot be formed. The positions of the primers and the probes for amplifying the japonica rice specific cpDNA fragment in the sequence are shown in FIG. 1.
All primers and probes (Table 1) were synthesized by Pidakon Biotechnology (Shanghai) Co., Ltd., and were diluted to 10. mu. mol/L with TE solution (pH 8.0) and stored at-20 ℃ for further use.
Primers and probes used in Table 1
5 extraction of DNA
Grinding rice seeds (rice) into powder; weighing 1.0g of the suspension into a 50 mL centrifuge tube, adding 5mL of CTAB lysate, and incubating overnight at 65 ℃; centrifuging at 15000rpm for 10min, and collecting supernatant; adding 2 times of CTAB precipitation solution, shaking, and standing at room temperature for 1 h; centrifuging at 15000rpm for 10min, collecting precipitate, adding 900 μ L1.2 mol/L sodium chloride solution, incubating at 65 deg.C, dissolving, and transferring into 2.0mL centrifuge tube; add 900. mu.L of chloroform: isoamyl alcohol (24: 1), vortex mixing evenly, and centrifuging for 10min at 12000 rpm; taking the supernatant, adding chloroform with the same volume, and centrifuging at 12000 rpm for 10 min; taking the supernatant, adding isopropanol with the volume 0.8 times that of the supernatant, turning upside down and uniformly mixing, and centrifuging at 12000 rpm for 10 min; taking the precipitate, adding 1mL 70% ethanol, vortexing, and centrifuging at 12000 rpm for 10 min; discarding the supernatant, taking the precipitate, and drying in a DNA concentrator; adding 200. mu.L of TE solution (pH 8.0), and incubating at 65 ℃ until the precipitate is completely dissolved; taking 2 mu LDNA solution to measure the concentration in an ultramicro nucleotide tester, diluting to 100 ng/mu L with TE solution (pH8.0), and storing at-20 ℃ for later use.
Optimization of 6-fold real-time fluorescent PCR reaction system
DNA of indica rice with cpDNA deletion type and japonica rice with cpDNA non-deletion type which are confirmed by sequencing are taken as templates, and the double real-time fluorescence PCR reaction system is optimized by using primers and probes for indica rice and japonica rice identification, and primers and probes for rice endogenous gos9 genes (shown in table 2). Reaction conditions are as follows: 35s at 95 ℃; 10s at 95 ℃, 40s at 60 ℃ and 40 cycles.
The optimization results (fig. 2-6) show that: only 1 amplification curve of the rice endogenous gos9 gene appears in the double real-time fluorescence PCR result of the cpDNA deletion type indica rice, and 2 amplification curves of the rice endogenous gos9 gene and japonica rice specific cpDNA fragment appear in the double real-time fluorescence PCR result of the cpDNA non-deletion type japonica rice; when the composition ratio of the primer and the probe of the rice endogenous gos9 gene to the primer and the probe of the japonica rice specific cpDNA fragment is 1:3 and 1:4, the fluorescence intensities of the rice endogenous gos9 gene and the japonica rice specific cpDNA fragment are all consistent, namely, both the rice endogenous gos9 gene and the japonica rice specific cpDNA fragment can be well amplified, but the ratio of 1:3 is selected in the aspect of saving the primer and the probe.
The finally established double real-time fluorescence PCR identification method of indica-japonica rice subspecies has the reaction system as follows: 2 XMix 10. mu.L, gos 9-F0.2. mu.L, gos 9-R0.2. mu.L, gos 9-P0.1. mu.L, rice-F40.6. mu.L, rice-R10.6. mu.L, rice-P0.3. mu.L, DNA (100 ng/. mu.L) 2. mu.L, ddH2O6.0. mu.L. The reaction conditions are as follows: 35s at 95 ℃; 10s at 95 ℃, 40s at 60 ℃ and 40 cycles.
TABLE 2 Duplex real-time fluorescent PCR optimized reaction System
TABLE 3 Ct values and fluorescence intensities for Duplex real-time fluorescent PCR
7. Compliance of authentication methods
376 parts of known indica type and japonica type rice seeds (rice) are identified by the duplex real-time fluorescence PCR method established above, and the results are shown in fig. 7 and fig. 8. All samples have obvious amplification curves of the endogenous gos9 genes of rice, the indica subspecies rice only has 1 amplification curve of the endogenous gos9 genes of rice, and the japonica subspecies rice seeds all have 2 amplification curves of the endogenous gos9 genes of rice and the japonica rice specific cpDNA. The identification result of the method is highly consistent with the indica rice and japonica rice types of rice. Among 125 japonica rice variety samples, 121 samples of 2 curves are detected, and only 4 samples of 1 rice endogenous gos9 gene amplification curve are shown, wherein the coincidence rate with japonica rice is 96.8%, and the non-coincidence rate is 3.24%. In 251 indica rice variety samples, only 220 samples of 1 rice endogenous gos9 gene amplification curve are detected, 31 samples of 2 curves are detected, the coincidence rate with indica rice is 88.0%, and the non-coincidence rate is 12.0%. According to the query of the national Rice Data center (Rice Data Base), only 31 varieties with 1 curve exist, and 9 varieties have non-deletion type maternal blood relationship of japonica Rice cpDNA. The reason for the non-conformity may be that the non-glutinous rice is traditionally misnamed, or the non-glutinous rice is used as a female parent and the non-glutinous rice is used as a male parent to carry out multi-generation backcross to form a non-glutinous rice variety with the cytoplasm of the non-glutinous rice, or vice versa. The method is used for detecting and identifying indica-japonica rice subspecies, and has high accuracy.
The method only detects one gene locus, is much simpler than other published detection methods based on nuclear DNA and chloroplast DNA, and has more to dozens of DNA loci. The primer pair for amplifying the insertion deletion molecular marker (InDel) site of 45 pairs of Chua stars et al (2006) is used for carrying out PCR detection on 93 varieties (49 indica types and 43 japonica types) of typical indica-japonica rice from 10 Asia countries, and the result shows that the accuracy of identifying indica rice or japonica rice varieties by 41 pairs of InDel primers is higher than 80%. Zhao Wei et al (2008) detected 25 parts of each of typical indica rice and japonica rice samples by using 34 pairs of InDel primers, and the accuracy rate was over 90%. Rong Sheng (2011) selects 98 parts of cultivated Asia rice and 125 parts of common wild rice in the primary China as materials, 5-segment high mutation sequences in chloroplast are sequenced, all the materials can be classified into 3 groups, wherein the group I mainly comprises japonica rice and common wild rice, the group II mainly comprises indica rice, the group III mainly comprises common wild rice, and typical indica-japonica rice types are not subjected to compound comparison. The method only detects one site, and the coincidence rate of japonica rice (rice) and indica rice (rice) in 376 samples respectively reaches 96.8 percent and 88 percent, which shows that the method is simple and efficient. In reports with similar methods, common PCR methods are adopted and electrophoresis is required. The method adopts a real-time fluorescence PCR method, can monitor the PCR process in real time, does not need electrophoresis, saves time, reduces the pollution of PCR products to the environment in the electrophoresis process, and improves the accuracy of results. The rice endogenous gos9 gene and the special cpDNA of japonica rice are combined to establish a double real-time fluorescence PCR method, so that false negative of the indica rice identification result is effectively eliminated, namely when the amplification curve of the special cpDNA of japonica rice does not appear, the reason that the DNA extraction effect is not good or substances inhibiting PCR exist in DNA can be eliminated, and the result is really the cpDNA deletion type. Therefore, the method has the advantages of simplicity, high efficiency, rapidness, accuracy and the like.
SEQUENCE LISTING
<110> Fuzhou customs technology center
<120> indica-japonica subspecies rice duplex real-time fluorescence PCR identification method based on chloroplast DNA difference
<130>6
<160>6
<170>PatentIn version 3.3
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ttagcctccc gctgcaga 18
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ttgaggatta ttccatgatt cc 22
Claims (3)
1. A dual real-time fluorescence PCR identification probe for indica-japonica subspecies rice based on chloroplast DNA difference is characterized in that the probe is rice-P and has the sequence as follows: 5 '-HEX-ATG CAA TAG AGA GCG AGT GG-BHQ 1-3'.
2. A dual real-time fluorescence PCR identification method of indica-japonica subspecies rice based on chloroplast DNA difference is characterized in that: reaction system: 2 XMix 10. mu.L, primer gos 9-F0.2. mu. L, gos 9-R0.2. mu.L, probe gos 9-P0.1. mu.L, primer rice-F40.6. mu.L, rice-R10.6. mu.L, probe rice-P0.3. mu.L, 100 ng/. mu.L DNA 2. mu.L, ddH2O6.0 μ L; reaction conditions are as follows: 35s at 95 ℃; 10s at 95 ℃, 40s at 60 ℃ and 40 cycles.
3. The dual real-time fluorescence PCR identification method of indica-japonica subspecies rice based on chloroplast DNA difference as claimed in claim 2, wherein the fluorescence PCR identification method comprises the following steps: the primer gos9-F is 5'-TTA GCC TCC CGC TGC AGA-3', gos9-R is 5'-AGA GTC CAC AAG TGC TCC CG-3', and the probe gos9-P is 5 '-FAM-CGG CAG TGT GGT TGG TTT CTTCGG-TAMRA-3'; rice-F4 is 5'-AAT CGC AAC CCC TTT CCG C-3', rice-R1 is 5'-TTG AGG ATTATT CCA TGA TTC C-3', and probe rice-P is 5 '-HEX-ATG CAA TAG AGA GCG AGT GG-BHQ 1-3'.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010022551A1 (en) * | 2008-08-26 | 2010-03-04 | 天津市农业科学院中心实验室 | A method for checking insect-resistant transgenic rice |
| CN103333956A (en) * | 2013-06-06 | 2013-10-02 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Assay primer and assay method of PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) of rice endogenous gene gos9 |
| CN104195225A (en) * | 2014-07-03 | 2014-12-10 | 武汉大学 | Quantitative PCR method for rapidly identifying transgenic paddy rice homozygote |
-
2020
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010022551A1 (en) * | 2008-08-26 | 2010-03-04 | 天津市农业科学院中心实验室 | A method for checking insect-resistant transgenic rice |
| CN103333956A (en) * | 2013-06-06 | 2013-10-02 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Assay primer and assay method of PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) of rice endogenous gene gos9 |
| CN104195225A (en) * | 2014-07-03 | 2014-12-10 | 武汉大学 | Quantitative PCR method for rapidly identifying transgenic paddy rice homozygote |
Non-Patent Citations (2)
| Title |
|---|
| 付伟等: "二重荧光定量PCR检测转基因大豆DAS81419", 《植物检疫》 * |
| 杨杰等: "江淮流域杂草稻叶绿体DNA的籼粳分化", 《中国水稻科学》 * |
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