CN108728561B - Method for identifying authenticity of blue vegetable No. 1 and special primer - Google Patents
Method for identifying authenticity of blue vegetable No. 1 and special primer Download PDFInfo
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Abstract
The invention discloses a method for identifying the authenticity of blue vegetable No. 1 and a special primer. The primer pair for identifying or assisting in identifying the cabbage No. 1 provided by the invention is a primer pair consisting of two single-stranded DNA molecules shown as a sequence 1 and a sequence 2 in a sequence table. Meanwhile, the invention also provides another 21 primer pairs for identifying or assisting in identifying the cabbage No. 1. Experiments prove that the primer pair provided by the invention can accurately identify the blue vegetable No. 1 and the cabbage type rape. The method has important significance for identifying the authenticity of the blue vegetable No. 1.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for identifying the authenticity of blue vegetable No. 1 and a special primer.
Background
The blue cabbage No. 1 is a two-heterology addition line which is obtained by carrying out somatic hybridization on isatis tinctoria and brassica napus and then carrying out continuous backcross with the rape and contains two same isatis tinctoria heterochromosomes and is subcultured by adopting a tissue culture method; the leaf of the strain shows a certain in vitro inhibition effect on influenza virus A/PR/8/34(H1N 1). The hybrid mutant is named as Lancai No. 1, and new species identification is carried out by the organization of a seed management station in Beijing City in 11 months and 2 days in 2014, and the serial number of a variety identification certificate is as follows: jingpin vegetable 2014032.
With the development of molecular biology, the molecular identification technology has become a beneficial supplement of the traditional identification method of traditional Chinese medicinal materials. The PCR method can be used for effectively and horizontally identifying various traditional Chinese medicinal materials in interspecies and under the interspecies, and compared with morphological observation and chemical component identification, the PCR method has more accurate identification result. At present, no method for identifying the authenticity of the blue vegetable No. 1 exists.
Disclosure of Invention
The invention aims to provide a method for identifying the authenticity of blue vegetable No. 1 and a special primer.
The primer pair for identifying or assisting in identifying the cabbage No. 1 provided by the invention is a primer pair consisting of two single-stranded DNA molecules shown as a sequence 1 and a sequence 2 in a sequence table.
The invention also provides a primer pair group for identifying or assisting in identifying the cabbage No. 1.
The primer pair group for identifying or assisting in identifying the cabbage No. 1 provided by the invention specifically comprises the primer pair of claim 1 and any one or more of the following primer pairs:
a primer pair 2 consisting of two single-stranded DNA molecules shown in a sequence 3 and a sequence 4 in a sequence table;
a primer pair 3 consisting of two single-stranded DNA molecules shown in a sequence 5 and a sequence 6 in a sequence table;
a primer pair 4 consisting of two single-stranded DNA molecules shown in a sequence 7 and a sequence 8 in a sequence table;
a primer pair 5 consisting of two single-stranded DNA molecules shown in a sequence 9 and a sequence 10 in a sequence table;
a primer pair 6 consisting of two single-stranded DNA molecules shown in a sequence 11 and a sequence 12 in a sequence table;
a primer pair 7 consisting of two single-stranded DNA molecules shown in a sequence 13 and a sequence 14 in a sequence table;
a primer pair 8 consisting of two single-stranded DNA molecules shown as a sequence 15 and a sequence 16 in a sequence table;
a primer pair 9 consisting of two single-stranded DNA molecules shown in a sequence 17 and a sequence 18 in a sequence table;
a primer pair 10 consisting of two single-stranded DNA molecules shown as a sequence 19 and a sequence 20 in a sequence table;
a primer pair 11 consisting of two single-stranded DNA molecules shown in a sequence 21 and a sequence 22 in a sequence table;
a primer pair 12 consisting of two single-stranded DNA molecules shown in a sequence 23 and a sequence 24 in a sequence table;
a primer pair 13 consisting of two single-stranded DNA molecules shown in a sequence 25 and a sequence 26 in a sequence table;
a primer pair 14 consisting of two single-stranded DNA molecules shown in a sequence 27 and a sequence 28 in a sequence table;
a primer pair 15 consisting of two single-stranded DNA molecules shown as a sequence 29 and a sequence 30 in a sequence table;
a primer pair 16 consisting of two single-stranded DNA molecules shown as a sequence 31 and a sequence 32 in a sequence table;
a primer pair 17 consisting of two single-stranded DNA molecules shown in a sequence 33 and a sequence 34 in a sequence table;
a primer pair 18 consisting of two single-stranded DNA molecules shown in a sequence 35 and a sequence 36 in a sequence table;
a primer pair 19 consisting of two single-stranded DNA molecules shown in a sequence 37 and a sequence 38 in a sequence table;
a primer pair 20 consisting of two single-stranded DNA molecules shown in a sequence 39 and a sequence 40 in a sequence table;
a primer pair 21 consisting of two single-stranded DNA molecules shown as a sequence 41 and a sequence 42 in a sequence table;
a primer pair 22 consisting of two single-stranded DNA molecules shown in a sequence 43 and a sequence 44 in a sequence table.
The invention also provides a kit for identifying or assisting in identifying the cabbage No. 1.
The kit for identifying or assisting in identifying the cabbage No. 1, provided by the invention, contains the primer pair or the primer pair group, dNTP and DNA polymerase.
The invention also provides a method for preparing the primer pair, a method for preparing the primer pair and a method for preparing the kit.
The method for preparing the primer pair provided by the invention can specifically comprise the step of respectively and independently packaging the two single-stranded DNA molecules forming the primer pair.
The method for preparing the primer pair group provided by the invention can specifically comprise the step of respectively and independently packaging two single-stranded DNA molecules forming each primer pair in the primer pair group.
The method for preparing the kit provided by the invention specifically comprises the following steps (A) or (B):
(A) packaging the two single-stranded DNA molecules forming the primer pair respectively and independently, and then packaging the two single-stranded DNA molecules and the dNTP and the DNA polymerase which are packaged respectively and independently in the same reagent box;
(B) and packaging the two single-stranded DNA molecules forming each primer pair in the primer pair group respectively and independently, and then packaging the two single-stranded DNA molecules and the dNTP and the DNA polymerase which are respectively packaged independently in the same reagent box.
The application of the primer pair or the primer pair group or the kit in identifying or assisting in identifying the cabbage No. 1 also belongs to the protection scope of the invention.
The invention provides a method for identifying or assisting in identifying whether a sample to be detected contains cabbage No. 1 or not and a method for identifying or assisting in identifying whether the sample to be detected is the cabbage No. 1 or the brassica napus.
The method for identifying or assisting in identifying whether the sample to be detected contains the cabbage No. 1 specifically comprises the following steps (A) or (B):
(A) the method for identifying or assisting in identifying whether the sample to be detected contains the cabbage No. 1 by using the primer pair (the sequence 1 and the sequence 2) comprises the following steps:
(a1) extracting genome DNA from a sample to be detected as a template, and performing PCR amplification by using the primer pair (sequence 1 and sequence 2);
(a2) determining whether the sample to be detected contains the cabbage No. 1 according to the size of the PCR product obtained in the step (a1) by the following method: if the PCR product contains a DNA fragment of 500-750bp (such as 542bp), the sample to be detected contains or is candidate to contain the cabbage No. 1; if the PCR product does not contain the DNA fragment of 500-750bp (such as 542bp), the sample to be detected does not contain or candidate does not contain the blue vegetable No. 1;
(B) the method for identifying or assisting in identifying whether the sample to be detected contains the cabbage No. 1 by using the primer pair group comprises the following steps:
(b1) extracting genome DNA from a sample to be detected as a template, and respectively carrying out PCR amplification by using each primer pair in the primer pair group of claim 2;
(b2) determining whether the sample to be detected contains the cabbage No. 1 according to the size of the PCR product obtained in the step (b1) by the following method: if the product obtained by performing PCR amplification on each primer pair in the primer pair group contains target DNA fragments with corresponding sizes, the sample to be detected contains or is candidate to contain the cabbage No. 1; otherwise, the sample to be detected does not contain or candidate does not contain the blue dish No. 1;
the size of the target DNA fragment obtained by PCR amplification of the primer pair 1 (sequence 1 and sequence 2) is 500-750bp (such as 542 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 2 (sequence 3 and sequence 4) is 750-1000bp (such as 832 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 3 (sequence 5 and sequence 6) is 500-750bp (such as 591 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 4 (sequence 7 and sequence 8) is 250-500bp (such as 260 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 5 (sequence 9 and sequence 10) is 750-1000bp (such as 790 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 6 (the sequence 11 and the sequence 12) is 500-750bp (such as 583 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 7 (sequence 13 and sequence 14) is 500-750bp (such as 538 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 8 (sequence 15 and sequence 16) is 750-1000bp (such as 855 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 9 (sequence 17 and sequence 18) is 100-250bp (such as 107 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 10 (sequence 19 and sequence 20) is 500-750bp (such as 661 bp);
the size of the target DNA fragment obtained by PCR amplification using the primer pair 11 (sequence 21 and sequence 22) is 750-1000bp (e.g., 823 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 12 (sequence 23 and sequence 24) is 500-750bp (such as 599 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 13 (sequence 25 and sequence 26) is 750-1000bp (such as 803 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 14 (sequence 27 and sequence 28) is 750-1000bp (such as 755 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 15 (sequence 29 and sequence 30) is 500-750bp (e.g. 539 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 16 (sequence 31 and sequence 32) is 500-750bp (such as 637 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 17 (sequence 33 and sequence 34) is 750-1000bp (such as 866 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 18 (sequence 35 and sequence 36) is 500-750bp (such as 682 bp);
the size of the target DNA fragment obtained by PCR amplification using the primer pair 19 (sequence 37 and sequence 38) is 750-1000bp (such as 840 bp);
the size of the target DNA fragment obtained by PCR amplification using the primer pair 20 (sequence 39 and sequence 40) is 750-1000bp (e.g. 772 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 21 (sequence 41 and sequence 42) is 750-1000bp (such as 775 bp);
the size of the target DNA fragment obtained by PCR amplification using the primer pair 22 (SEQ ID NO: 43 and SEQ ID NO: 44) is 100-250bp (e.g., 156 bp).
The method for identifying or assisting in identifying whether the sample to be detected is brassica napus No. 1 or brassica napus provided by the invention specifically comprises the following steps (A) or (B):
(A) the method for identifying or assisting in identifying whether the sample to be detected is the brassica 1 or the brassica napus by using the primer pair (the sequence 1 and the sequence 2) comprises the following steps:
(a1) extracting genome DNA from a sample to be detected as a template, and performing PCR amplification by using the primer pair (sequence 1 and sequence 2);
(a2) determining whether the sample to be detected is the blue vegetable No. 1 or the cabbage type rape according to the size of the PCR product obtained in the step (a1) by the following method: if the PCR product contains a DNA fragment of 500-750bp (such as 542bp), the sample to be detected is or is selected as the blue vegetable No. 1; if the PCR product does not contain the DNA fragment of 500-750bp (such as 542bp), the sample to be detected is or is selected as cabbage type rape;
(B) the method for identifying or assisting in identifying whether the sample to be detected is the brassica napus No. 1 or the brassica napus by using the primer pair group comprises the following steps:
(b1) extracting genome DNA from a sample to be detected as a template, and respectively carrying out PCR amplification on each primer pair in the primer pair group;
(b2) determining whether the sample to be detected is the blue vegetable No. 1 or the cabbage type rape according to the size of the PCR product obtained in the step (b1) by the following method: if the product obtained by performing PCR amplification on each primer pair in the primer pair group contains target DNA fragments with corresponding sizes, the sample to be detected is or is candidate to be the cabbage No. 1; otherwise, the sample to be detected is or is selected as the cabbage type rape;
the size of the target DNA fragment obtained by PCR amplification of the primer pair 1 (sequence 1 and sequence 2) is 500-750bp (such as 542 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 2 (sequence 3 and sequence 4) is 750-1000bp (such as 832 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 3 (sequence 5 and sequence 6) is 500-750bp (such as 591 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 4 (sequence 7 and sequence 8) is 250-500bp (such as 260 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 5 (sequence 9 and sequence 10) is 750-1000bp (such as 790 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 6 (the sequence 11 and the sequence 12) is 500-750bp (such as 583 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 7 (sequence 13 and sequence 14) is 500-750bp (such as 538 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 8 (sequence 15 and sequence 16) is 750-1000bp (such as 855 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 9 (sequence 17 and sequence 18) is 100-250bp (such as 107 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 10 (sequence 19 and sequence 20) is 500-750bp (such as 661 bp);
the size of the target DNA fragment obtained by PCR amplification using the primer pair 11 (sequence 21 and sequence 22) is 750-1000bp (e.g., 823 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 12 (sequence 23 and sequence 24) is 500-750bp (such as 599 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 13 (sequence 25 and sequence 26) is 750-1000bp (such as 803 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 14 (sequence 27 and sequence 28) is 750-1000bp (such as 755 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 15 (sequence 29 and sequence 30) is 500-750bp (e.g. 539 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 16 (sequence 31 and sequence 32) is 500-750bp (such as 637 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 17 (sequence 33 and sequence 34) is 750-1000bp (such as 866 bp);
the size of the target DNA fragment obtained by PCR amplification by using the primer pair 18 (sequence 35 and sequence 36) is 500-750bp (such as 682 bp);
the size of the target DNA fragment obtained by PCR amplification using the primer pair 19 (sequence 37 and sequence 38) is 750-1000bp (such as 840 bp);
the size of the target DNA fragment obtained by PCR amplification using the primer pair 20 (sequence 39 and sequence 40) is 750-1000bp (e.g. 772 bp);
the size of the target DNA fragment obtained by PCR amplification of the primer pair 21 (sequence 41 and sequence 42) is 750-1000bp (such as 775 bp);
the size of the target DNA fragment obtained by PCR amplification using the primer pair 22 (SEQ ID NO: 43 and SEQ ID NO: 44) is 100-250bp (e.g., 156 bp).
In the method, the sample to be detected is brassica oleracea No. 1 or brassica napus.
In both methods, the annealing temperature used for PCR amplification is 50-60 ℃. The specific annealing temperatures for each primer are shown in Table 1 in example 1.
Further, the specific PCR amplification procedure employed was: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, 50-60 ℃ (see table 1 in example 1 for specific annealing temperatures for each primer) annealing for 30s, extension at 72 ℃ for 60s, 35 cycles; extension at 72 ℃ for 5 min.
In both methods, the final concentration of the upstream and downstream primers in the reaction system was 0.4. mu.M during PCR amplification.
In practical application, whether the PCR product contains the target DNA fragment or not is judged, the PCR product can be detected by carrying out agarose gel electrophoresis, and if the electrophoresis result shows that the PCR product contains the target band, the PCR product contains the target DNA fragment; otherwise, the target DNA fragment is not contained. Of course, the determination of whether the PCR product contains the DNA fragment of interest is also carried out by a sequencing method.
Experiments prove that the primers provided by the invention can be used for accurately and quickly identifying the blue vegetable No. 1 and the cabbage type rape by a PCR method. The method has important significance for identifying the authenticity of the blue vegetable No. 1.
Drawings
FIG. 1 is a diagram showing the result of PCR electrophoresis using 22 pairs of primers. M is DL2000Marker (each band is 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from large to small in sequence; 1 is isatin, 2 is brassica napus; 3 is brassica oleracea number 1; the Arabic numerals above each small electrophoretogram in the figure represent the electropherogram of the amplification product corresponding to the primer pair with the same Arabic numeral in the primer pair number in Table 1.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Blue dish No. 1: the inventor of 'Yangjian, Yuan, Tangjinfu and the like, characteristic features of the cabbage No. 1 and a standardized cultivation technology, modern Chinese medicine 2016,18(8):1006 and 1008', the public can obtain from the applicant and can only be used in repeated experiments.
Example 1 identification of blue vegetable No. 1
First, experimental material, instrument and reagent
1. Experimental Material
Sterile plant of Isatis tinctoria, Brassica napus and Brassica oleracea No. 1 cultured in laboratory.
2. Laboratory apparatus
GeneAmp model 9700 PCR amplification apparatus (Applied biosystems); model DYY-12 computerized triple-purpose electrophoresis apparatus (Beijing Liuyi instruments factory); HE99X-15-1.5 type electrophoresis tank (Hoefer Corp.); SYNGENE gel imaging system (GENE corporation); ND-2000 model nucleic acid/protein Analyzer (Nanodrop Co.); model Geno grind 2010 refrigerated mixer ball mill (RETSCH).
3. Experimental reagent
Agarose was purchased from Biowest; ethidium bromide was purchased from Amresco; rTaq DNA polymerase, DL2000 DNAmarker was purchased from TaKaRa; other reagents are all domestic analytical purifiers.
Second, Experimental methods
1. Design of blue vegetable No. 1 specific identifying primer
Genome re-sequencing is carried out on blue vegetable No. 1 plants by the Baimaike company, genomes of the cabbage type rape are obtained by downloading from NCBI, the blue vegetable No. 1 and the genomes of the cabbage type rape are compared by using software BLAST, and a unique sequence of the blue vegetable No. 1 different from the cabbage type rape is selected. Primer design was performed using software Primer premier 5.0 based on these unique sequences. A total of 22 primer pairs were obtained, and the specific primer sequences are shown in Table 1.
TABLE 1 primer sequences
2. Genomic DNA extraction
Taking 100mg fresh weight of experimental material, grinding by using liquid nitrogen, extracting total DNA from the obtained powder by using a CTAB method, and detecting the concentration and purity of the DNA by using a nucleic acid detector.
3. PCR amplification
The concentration of the genomic DNA obtained in step 2 was diluted to 20 ng. mu.L-1And storing at-20 deg.C for use. And selecting identification primers to amplify the sample No. 1 of the isatis indigotica fort, the rape and the blue vegetable. The primer sequences are shown in Table 1. The PCR amplification system (25. mu.L) was: 10 XPCR Buffer 2.5. mu.L, rTaqDNA polymerase 0.2. mu.L, dNTPs 2. mu.L, 10 pmol. mu.L-1mu.L of each of the upstream and downstream primers of (1), 1. mu.L of the template, and a total of 25. mu.L of sterilized distilled water. The PCR amplification procedure was: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, 50-60 ℃ (specific annealing temperature for each primer pair see table 1) annealing for 30s, extension at 72 ℃ for 60s, 35 cycles; extension at 72 ℃ for 5 min.
The PCR products were detected by electrophoresis on a 1% agarose gel, observed and recorded on a gel imaging system.
Third, experimental results
The results are shown in FIG. 1. As can be seen, specific bands were amplified with 22 pairs of primers in both Isatis tinctoria and Isatis tinctoria No. 1, and the band sizes were consistent with the corresponding amplified fragment sizes in Table 1. While there is no corresponding band in brassica napus. Therefore, the 22 pairs of primers can be used for identifying the brassica napus No. 1 and the brassica napus.
<110> institute of traditional Chinese medicine of Chinese academy of traditional Chinese medicine
Method for identifying authenticity of <120> blue vegetable No. 1 and special primer
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<170> PatentIn version 3.5
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<220>
<223>
<400> 33
gaatgaagaa cacggtagca aataagaa 28
<210> 34
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 34
ttaagattac acgccgacct aatgac 26
<210> 35
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 35
cggtggtgac agaagtgatg cctat 25
<210> 36
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 36
tgcaatccaa gaataatgtc ggagc 25
<210> 37
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 37
agaccataaa ccctccaagc ctcaa 25
<210> 38
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 38
cctcgccaat cctacttaca tccac 25
<210> 39
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 39
ctgttcctgt tgctcctatc ttt 23
<210> 40
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 40
ttctgctgga tttagaccat ttc 23
<210> 41
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 41
gaaatagtct gtgggcgtct gtgct 25
<210> 42
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 42
gtatctcctc ctgccaggct cgtat 25
<210> 43
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 43
ttaagcctac acctcaacct ccag 24
<210> 44
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 44
ttggttccat ccattggagt agtt 24
Claims (5)
1. The primer pair for identifying the cabbage No. 1 is characterized by comprising the following components in parts by weight: the primer pair is composed of two single-stranded DNA molecules shown in a sequence 1 and a sequence 2 in a sequence table.
2. The primer pair group for identifying the blue vegetable No. 1 is characterized in that: the primer pair group is composed of the primer pair of claim 1 and any one or more of the following primer pairs:
a primer pair 2 consisting of two single-stranded DNA molecules shown in a sequence 3 and a sequence 4 in a sequence table;
a primer pair 3 consisting of two single-stranded DNA molecules shown in a sequence 5 and a sequence 6 in a sequence table;
a primer pair 4 consisting of two single-stranded DNA molecules shown in a sequence 7 and a sequence 8 in a sequence table;
a primer pair 5 consisting of two single-stranded DNA molecules shown in a sequence 9 and a sequence 10 in a sequence table;
a primer pair 6 consisting of two single-stranded DNA molecules shown in a sequence 11 and a sequence 12 in a sequence table;
a primer pair 7 consisting of two single-stranded DNA molecules shown in a sequence 13 and a sequence 14 in a sequence table;
a primer pair 8 consisting of two single-stranded DNA molecules shown as a sequence 15 and a sequence 16 in a sequence table;
a primer pair 9 consisting of two single-stranded DNA molecules shown in a sequence 17 and a sequence 18 in a sequence table;
a primer pair 10 consisting of two single-stranded DNA molecules shown as a sequence 19 and a sequence 20 in a sequence table;
a primer pair 11 consisting of two single-stranded DNA molecules shown in a sequence 21 and a sequence 22 in a sequence table;
a primer pair 12 consisting of two single-stranded DNA molecules shown in a sequence 23 and a sequence 24 in a sequence table;
a primer pair 13 consisting of two single-stranded DNA molecules shown in a sequence 25 and a sequence 26 in a sequence table;
a primer pair 14 consisting of two single-stranded DNA molecules shown in a sequence 27 and a sequence 28 in a sequence table;
a primer pair 15 consisting of two single-stranded DNA molecules shown as a sequence 29 and a sequence 30 in a sequence table;
a primer pair 16 consisting of two single-stranded DNA molecules shown as a sequence 31 and a sequence 32 in a sequence table;
a primer pair 17 consisting of two single-stranded DNA molecules shown in a sequence 33 and a sequence 34 in a sequence table;
a primer pair 18 consisting of two single-stranded DNA molecules shown in a sequence 35 and a sequence 36 in a sequence table;
a primer pair 19 consisting of two single-stranded DNA molecules shown in a sequence 37 and a sequence 38 in a sequence table;
a primer pair 20 consisting of two single-stranded DNA molecules shown in a sequence 39 and a sequence 40 in a sequence table;
a primer pair 21 consisting of two single-stranded DNA molecules shown as a sequence 41 and a sequence 42 in a sequence table;
a primer pair 22 consisting of two single-stranded DNA molecules shown in a sequence 43 and a sequence 44 in a sequence table.
3. The kit for identifying the cabbage No. 1 is characterized by comprising the following components in parts by weight: the kit contains the primer pair of claim 1 or the primer pair of claim 2, dNTP and DNA polymerase.
4. Use of the primer pair of claim 1 or the primer pair set of claim 2 or the kit of claim 3 for identifying whether a sample to be tested is brassica napus No. 1 or brassica napus;
the sample to be detected is cabbage No. 1 or cabbage type rape.
5. The method for identifying whether a sample to be tested is brassica napus No. 1 or brassica napus by using the primer pair group of claim 2, comprising the steps of:
(b1) extracting genome DNA from a sample to be detected as a template, and respectively carrying out PCR amplification by using each primer pair in the primer pair group of claim 2;
(b2) determining whether the sample to be detected is the blue vegetable No. 1 or the cabbage type rape according to the size of the PCR product obtained in the step (b1) by the following method: if the product obtained by performing PCR amplification on each primer pair in the primer pair group contains target DNA fragments with corresponding sizes, the sample to be detected is the cabbage No. 1; otherwise, the sample to be detected is the cabbage type rape;
the size of a target DNA fragment obtained by PCR amplification by using the primer pair of claim 1 is 542 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 2 is 832 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 3 is 591 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 4 is 260 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 5 is 790 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 6 is 583 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 7 is 538 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 8 is 855 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 9 is 107 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 10 is 661 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 11 is 823 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 12 is 599 bp;
the size of a target DNA fragment obtained by PCR amplification of the primer pair 13 is 803 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 14 is 755 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 15 is 539 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the 16 primer pair is 637 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 17 is 866 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 18 is 682 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 19 is 840 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 20 is 772 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 21 is 775 bp;
the size of a target DNA fragment obtained by carrying out PCR amplification on the primer pair 22 is 156 bp;
the sample to be detected is cabbage No. 1 or cabbage type rape.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710240063.8A CN108728561B (en) | 2017-04-13 | 2017-04-13 | Method for identifying authenticity of blue vegetable No. 1 and special primer |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710240063.8A CN108728561B (en) | 2017-04-13 | 2017-04-13 | Method for identifying authenticity of blue vegetable No. 1 and special primer |
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| Publication Number | Publication Date |
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| CN108728561A CN108728561A (en) | 2018-11-02 |
| CN108728561B true CN108728561B (en) | 2021-06-08 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450938A (en) * | 2014-12-25 | 2015-03-25 | 中国中医科学院中药研究所 | Ginseng identification method and special kit |
| CN104805097A (en) * | 2014-01-23 | 2015-07-29 | 中国人民解放军第二军医大学 | Coding sequences and applications of isatis indigotica fortune pinoresinol reductase protein |
| CN105230486A (en) * | 2015-09-29 | 2016-01-13 | 华中农业大学 | Breeding method of cabbage type rape woad oil cytoplasm male sterile line |
| CN106148355A (en) * | 2016-06-30 | 2016-11-23 | 中国人民解放军第二军医大学 | The application in regulation and control Lignanoids compounds synthesis of the Isatis indigotica Fort. IiAP2/ERF049 gene |
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2017
- 2017-04-13 CN CN201710240063.8A patent/CN108728561B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805097A (en) * | 2014-01-23 | 2015-07-29 | 中国人民解放军第二军医大学 | Coding sequences and applications of isatis indigotica fortune pinoresinol reductase protein |
| CN104450938A (en) * | 2014-12-25 | 2015-03-25 | 中国中医科学院中药研究所 | Ginseng identification method and special kit |
| CN105230486A (en) * | 2015-09-29 | 2016-01-13 | 华中农业大学 | Breeding method of cabbage type rape woad oil cytoplasm male sterile line |
| CN106148355A (en) * | 2016-06-30 | 2016-11-23 | 中国人民解放军第二军医大学 | The application in regulation and control Lignanoids compounds synthesis of the Isatis indigotica Fort. IiAP2/ERF049 gene |
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| Computational identification and systematic classification of novel GRAS genes in Isatis indigotica;Zhang L等;《Chin J Nat Med》;20160331;第14卷(第3期);第161-176页 * |
| 蓝菜1号特征特性及标准化栽培技术;杨健等;《中国现代中药》;20160831;第18卷(第8期);第1006-1008页 * |
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