CN103834737B - Transgenic rice Kefeng No.2 line specific PCR detection primer as well as qualitative detection method and kit - Google Patents
Transgenic rice Kefeng No.2 line specific PCR detection primer as well as qualitative detection method and kit Download PDFInfo
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Abstract
The invention discloses a transgenic rice Kefeng No.2 line specific polymerase chain reaction (PCR) detection primer as well as a qualitative detection method and a kit. Nucleotide sequences of the specific detection primer are respectively represented in SEQ ID No. 7 and SEQ ID No. 8. Furthermore, the invention discloses a PCR qualitative detection method for specificity of a transgenic rice Kefeng No.2 line, wherein the method comprises the steps of (1) extracting DNA of a rice sample to be detected; (2) establishing a PCR reaction system through the specific detection primer by taking the extracted DNA as a template, and amplifying; and (3) regarding that the rice sample to be detected is transgenic rice Kefeng No.2 if about 201bp of strips are amplified from the sample to be detected; or regarding that the rice sample to be detected is not transgenic rice Kefeng No.2 if about 201bp of strips are not amplified. Specificity and sensitivity experiments show that the PCR qualitative detection method disclosed by the invention is strong in specificity and high in sensitivity.
Description
Technical field
The present invention relates to the specific PCR of transgenic rice lines and detect primer and detection kit, particularly relate to the rich No. 2 strain specificity PCR of transgenic paddy rice section and detect primer, qualitative checking method and detection kit, belong to transgenic rice lines specific detection field.
Background technology
Along with the extensive plantation of transgenic plant, the safety issue of genetically modified food also causes people to pay close attention to greatly, and existing more than 30 country implements transgenic product mark system in succession, comprises China.The enforcement of transgenic labeling system depends on the composition detection to transgenic plant and converted products thereof.Round pcr detects most widely used method in transgenic product both at home and abroad at present.When applying this technology and carrying out genetically modified crops qualitative detection, according to its specificity, be divided into selective mechanisms method, gene specific method, built specificity method and strain specificity method.Event-specific detection is that the joining region sequence by detecting insertion vector and Plant Genome realizes, due to each Transgenic Plant Lines, all there is the joining region sequence of special exogenous insertion vector and Plant Genome, compare with first three methods, strain specificity has higher specificity and accuracy, the most applicablely does GMO detection.
At present, partial monopoly and the bibliographical information event-specific detection method of transgenic plant is had.Such as: magnify the people such as soldier established transgenosis MON863 corn event-specific detection method in 2006; The people such as Xie Jiajian establish a series of event-specific detection method such as transgenic paddy rice Kemingdao, Bt Shanyou 63, section rich No. 6 and rich No. 8 of section etc.; The people such as Lu Changming established the event-specific detection method of a series of transgene rape in 2007.But, so far, not yet find any strain specificity PCR detection method about rich No. 2 of transgenic paddy rice section.
Summary of the invention
An object of the present invention is to provide a pair and detects primer for the strain specificity PCR detecting rich No. 2 of transgenic paddy rice section;
Two of object of the present invention sets up the strain specificity PCR qualitative checking method of rich No. 2 of a kind of transgenic paddy rice section;
Three of object of the present invention is to provide the strain specificity PCR qualitative detection test kit of rich No. 2 of a kind of transgenic paddy rice section.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The PCR that the present invention provide firstly for detecting rich No. 2 strain specificities of transgenic paddy rice section detects primer, and described primer is selected from any pair in following 5 pairs of primers: nucleotides sequence is classified as the primer pair 1 shown in SEQID No.3 and SEQ ID No.4; Nucleotides sequence is classified as the primer pair 2 shown in SEQ ID No.5 and SEQ ID No.6; Nucleotides sequence is classified as primer pair 3 shown in SEQ ID No.7 and SEQ ID No.8; Nucleotides sequence is classified as the primer pair 4 shown in SEQ ID No.9 and SEQ ID No.10; Nucleotides sequence is classified as the primer pair 5 shown in SEQ ID No.11 and SEQ ID No.12; Preferably, described PCR detects primer is the primer pair shown in SEQ ID No.7 and SEQ ID No.8.
The present invention is that SCK gene (SEQ ID No.1) obtains external source insertion gene 5 ' end paddy rice flanking sequence and 3 ' end paddy rice flanking sequence by Hi-TAIL PCR according to the goal gene inserted in rich No. 2 strains of transgenic paddy rice section, and the exogenous insertion vector containing SCK gene obtained and both sides 5 ' thereof are held and the joining region nucleotides sequence of 3 ' side wing rice sequences is classified as shown in SEQ ID No.2; 5 pairs of specificity of transformant primers are designed according to the joining region nucleotide sequence that rich No. 2 exogenous insertion vectors of section and 3 ' end paddy rice flanking sequence are formed, the nucleotide sequence of 5 pairs of primers is respectively shown in SEQ ID No.3-SEQ IDNo.12, application PCR reaction conditions, adopt 5 pairs of primers to carry out pcr amplification respectively, 2 repetitions are set; From amplification, these 5 pairs of primers all can amplify expection fragment specifically from rich No. 2 samples of section, but the most clear with the band of the primer amplification shown in SEQ ID No.7 and SEQ ID No.8.Therefore, the present invention preferably adopts primer shown in SEQ ID No.7 and SEQ ID No.8 to detect primer as the strain specificity PCR detecting rich No. 2 of transgenic paddy rice section.
Two of object of the present invention is to provide the strain specificity PCR detection method of rich No. 2 of a kind of transgenic paddy rice section, and the method comprises: (1) extracts the DNA of paddy rice sample to be detected; (2) with the DNA extracted for template, set up PCR amplification system with Nucleotide shown in SEQ ID No.7 and SEQ ID No.8 for amplification upstream and downstream primer and to go forward side by side performing PCR amplification; (3) if amplify the band of about 201bp from sample to be detected, then paddy rice sample to be detected is rich No. 2 of transgenic paddy rice section; If fail to amplify the band of about 201bp from sample to be detected, then rich No. 2 of paddy rice sample Bu Shi transgenic paddy rice section to be detected.
Wherein, the described method extracting DNA from paddy rice sample to be detected can be the various common methods extracting DNA from vegetable material, such as, can be the various methods such as CTAB method, guanidine isothiocyanate method or guanidine hydrochloride method.
In PCR reaction system, the final concentration of primer and annealing temperature all have a certain impact for the specificity sensitivity of detected result; The present invention has investigated the impact for the specificity detected and sensitivity of the final concentration of primer in PCR reaction system and annealing temperature, experimental result find primer final concentration be 0.2 μM, annealing temperature be 58 DEG C time, the specificity of detected result is the strongest, and sensitivity is the highest.
PCR amplification system described in the present invention can be set up with reference to following methods: the cumulative volume of reaction system is 25.0 μ L, wherein, 10 × PCR damping fluid 2.5 μ L, 25mmol/L magnesium chloride solution 1.5 μ L, dNTPs mixing solutions (each 2.5mmol/L) 2 μ L, 0.2 μm of ol/L upstream primer 0.5 μ L, 0.2 μm of ol/L downstream primer 0.5 μ L, 0.025U/ μ L Taq enzyme 1.5 μ L, 25mg/L DNA profiling 2.0 μ L, surplus is distilled water.
Described pcr amplification condition optimization is: 94 DEG C, 5min; 94 DEG C, 30s, 58 DEG C, 30s, 72 DEG C, 30s, 35 circulations; 94 DEG C, 2min.
Further aim of the present invention is to provide the strain specificity PCR qualitative detection test kit of rich No. 2 of a kind of transgenic paddy rice section, comprising: 10 × PCR damping fluid, magnesium chloride solution, dNTPs mixing solutions, detects upstream and downstream primer pair, Taq enzyme and distilled water; Wherein, the nucleotide sequence of described detection upstream and downstream primer pair is respectively shown in SEQ ID No.7 and SEQ ID No.8.
The strain specificity PCR qualitative checking method of rich No. 2 of transgenic paddy rice section adopting the present invention to set up rich No. 6 to the excellent section of transgenic paddy rice Π, Shan excellent 10, Shanyou 63, Anhui 80-4B, anti-excellent 97, extensive No. 1 of China, rich No. 2 of section, conventional rice bright extensive 63 and other transgenic plant are (such as: MON863 corn, GHB614 cotton, MON89788 soybean and modified rape GT 73 etc.) carry out qualitative detection, qualitative PCR amplification shows, in rich No. 2 rice genomes of section, amplification obtains the band (201bp) of object clip size, rich No. 6 of the excellent section of other transgenic paddy rice Π, Shan excellent 10, Shanyou 63, Anhui 80-4B, anti-excellent 97, China extensive No. 1 and other transgenic plant (MON863 corn, GHB614 cotton, MON89788 soybean and modified rape GT 73) and the genome such as conventional rice bright extensive 63 in do not have to increase and obtain the fragment of object clip size, consistent with object amplified fragments sequence by sequencing analysis show to increase in rich No. 2 rice genomes of the section sequence that obtains.Experimental result shows, the rich No. 2 strain specificity qualitative PCR detection method high specificities of the transgenic paddy rice section that the present invention sets up.
Sensitivity experiment result shows, the rich No. 2 paddy rice compositions of section are 1%, 0.5%, 0.1%, all amplify the band of object clip size in the PCR reaction of 0.05%, illustrated that the sensitivity of the rich No. 2 rice strain specific PCR qualitative checking methods of section of the present invention is 0.05%, there is the susceptibility of height.
Accompanying drawing explanation
Fig. 1 Hi-TAIL pcr amplification result; M:1Kb plus Marker1: PCR primer 2 is taken turns in downstream second: downstream third round PCR primer; 3: PCR primer 4 is taken turns in upstream second: upstream third round product.
Fig. 2 upstream and downstream order-checking splicing result (2614bp).
Fig. 3 is from the nested primers of the two ends design Hi-TAIL PCR of known array.
Fig. 4 Hi-TAIL pcr amplification result; PCR primer is taken turns in M:1Kb plus Marker1, upstream second; 2, upstream third round product; Note: downstream primer is without band.
Fig. 5 upstream and downstream order-checking splicing result.
Fig. 6 pcr amplification result; M:1Kb plus Marker1: blank; 2: bright extensive 86 negative controls 3: rich No. 2 of section.
The integrated structure that the checking of Fig. 7 long segment obtains; 1., 2., 3., 4., 5. represent respectively: increase from illustrated Position Design long segment primer.
Figure 81 primer amplification result: M:1Kb plus Marker; 1: blank 2: bright extensive 86 negative controls 3: rich No. 2 of section.
Figure 92 primer amplification result: M:100bp Marker; 1: blank 2: bright extensive 86 negative controls 3: rich No. 2 of section.
Figure 103 primer amplification result: M:1Kb plus Marker, 1: blank 2: bright extensive 86 negative controls 3: rich No. 2 of section.
Figure 114 primer amplification result: M:1Kb plus Marker, 1: blank 2: bright extensive 86 negative controls 3: rich No. 2 of section.
Figure 125 primer amplification result: M:1Kb plus Marker, 1: blank 2: bright extensive 86 negative controls 3: rich No. 2 of section.
Rich No. 2 transformant 5 ' of Figure 13 section hold flanking sequence Auele Specific Primer the selection result.
Figure 145 ' holds the specificity verification result of flanking sequence primer;
A:M:100bp Marker; 1: blank; 2: bright extensive 86 negative controls; 3: rich No. 2 positive controls of section; 4-19 is respectively: bright extensive 63, Anhui 21-B, middlely spend 11, excellent bright extensive 86, extensive No. 1 of the China of Shanyou 63, rich No. 6 of section, rich No. 6 of the excellent section of II, II, Shan are excellent 10, bar68-1, Anhui 80-4B, anti-excellent 97, eastern agriculture, Liaojing 371, loose round-grained rice 6; B:M:100bp Marker; 1: blank; 2: bright extensive 86 negative controls; 3: rich No. 2 positive controls of section; 4-7 is respectively: corn feminine gender, MON863, Bt176, MON810; 8-10 is respectively: cotton feminine gender, LL25, MON15985; 11-13 is respectively: soybean feminine gender, MON89788,40-3-2; 14,15 be respectively: rape T45, MS1; 16,17 be respectively: beet GT73, H7-1.
Rich No. 2 transformant 3 ' of Figure 15 section hold flanking sequence Auele Specific Primer the selection result.
The final concentration of primer and annealing temperature optimum result in Figure 16 PCR reaction system; The final concentration of primer is 0.1 μM/L; M:100bp Marker1: bright extensive 86 negative controls, 2: the rich No. 2 a:54 DEG C b:56 DEG C c:58 DEG C d:60 DEG C of e:62 DEG C of section.
The final concentration of primer and annealing temperature optimum result in Figure 17 PCR reaction system; The final concentration of primer is 0.2 μM/L; M:100bp Marker1: bright extensive 86 negative controls; 2: the rich No. 2 a:54 DEG C b:56 DEG C c:58 DEG C d:60 DEG C of e:62 DEG C of section.
The final concentration of primer and annealing temperature optimum result in Figure 18 PCR reaction system; The final concentration of primer is 0.4 μM/L; M:100bp Marker1: bright extensive 86 negative controls; 2: rich No. 2 of section; A:54 DEG C b:56 DEG C c:58 DEG C d:60 DEG C of e:62 DEG C.
The final concentration of primer and annealing temperature optimum result in Figure 19 PCR reaction system; The final concentration of primer is 0.8 μM/L; M:100bp Marker1: bright extensive 86 negative controls; 2: the rich No. 2 a:54 DEG C b:56 DEG C c:58 DEG C d:60 DEG C of e:62 DEG C of section.
The rich No. 2 transformant method specific detection of Figure 20 section; M:100bp Marker1: blank 2: bright extensive 86 negative controls 3: rich No. 2 positive controls of section; 4-19 is respectively: bright extensive 63, Anhui 21-B, middlely spend 11, excellent bright extensive 86, extensive No. 1 of the China of Shanyou 63, rich No. 6 of section, rich No. 6 of the excellent section of II, II, Shan are excellent 10, bar68-1, Anhui 80-4B, anti-excellent 97, eastern agriculture, Liaojing 371, loose round-grained rice 6.
The rich No. 2 transformant method specific detection of Figure 21 section; M:100bp Marker1: blank 2: bright extensive 86 negative controls, 3: rich No. 2 positive controls of section, 4-7 is respectively: corn feminine gender, MON863, Bt176, MON810; 8-10 is respectively: cotton feminine gender, LL25, MON15985; 11-13 is respectively: soybean feminine gender, MON89788,40-3-2; 14,15 be respectively: rape T45, MS1; 16,17 be respectively: beet GT73, H7-1.
The rich No. 2 transformant method sensitivity test results of Figure 22 section; M:100bp Marker1: blank 2, negative control 3,4,5,6,7, positive 100%, 1%, 0.5%, 0.1%, 0.05%.
Figure 23 detectability test result; M:100bp Marker A: blank B: negative control; 1-10: 10 parallel 11-20 of positive 0.1%: 10 of positive 0.05% is parallel; 21-30: 10 of positive 0.01% is parallel.
In rich No. 2 strains of embodiment 1 transgenic paddy rice section, external source inserts the acquisition of the flanking sequence of SCK gene
It is SCK gene that the rich No. 2 strain external sources of transgenic paddy rice section insert goal gene, and 415bp altogether, its base sequence is for shown in SEQ ID No.1.
Integrated structure (external source insertion gene flanking sequence) is obtained by Hi-TAIL PCR.
1, the first step: the nested primers designing Hi-TAIL PCR on goal gene SCK
1. downstream primer:
Sck-sp1CTTTCTCATCATCTTCATCCCTGGACTTG
Sck-sp2ACGATGGACTCCAGTCCGGCCGATTTGCAAGCCGAGTGACACGAATT
Sck-sp3TTGATTTAGTGCAGATGCATGAATCGC
Upstream primer:
Sck-Ap1GCACCATCTTCTTTGCTCTCTTTCTCTGT
Sck-Ap2ACGATGGACTCCAGTCCGGCCTTTCGTTTTAAAGGTGTGTGTGCTGGTAC
Sck-Ap3TGATGACTCAAGCGATGAACCTTCTGAG
Random primer:
AD1-1ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA
AD1-2ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT
AD1-3ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA
AD1-4ACGATGGACTCCAGAGCGGCCGCBDNBNNNAGGT
AD-C ACGATGGACTCCAGAG
2. PCR the results are shown in Figure 1.
3. upstream and downstream order-checking splicing result (2614bp) is shown in Fig. 2.
2, second step: the nested primers establishing Hi-TAIL PCR from the two ends of known array
The schematic diagram that Fig. 3 designs for nested primers.
1. downstream primer:
OSP-SP1AGATTAAAATAGCTTTCCCCCGTTGTAGC
OSP-SP2ACGATGGACTCCAGTCCGGCCCAAAGTGCTATCCACGATCCATAGCAAG
OSP-SP3CAATAGTCTCCACACCCCCCCACTATC
Upstream primer:
hptII-AP1GCGACGTCTGTCGAGAAGTTTCTGATC
hptII-AP2ACGATGGACTCCAGTCCGGCCAGGGCGAAGAATCTCGTGCTTTCAG
hptII-AP3CGTTATGTTTATCGGCACTTTGCATCG
Random primer: the same
2. PCR the results are shown in Figure 4.
3. upstream and downstream order-checking splicing result (3840bp) is shown in Fig. 5.
3. the 3rd step: the rice sequences obtained according to 5 ' end, obtains 3 ' end rice sequences.Then, rice starter establishes upstream primer, paddy rice 3 ' terminal sequence establishes downstream primer, obtain 3 ' end flanking sequence by long segment amplification.
1. downstream primer:
OSJ-3'-AP：CGCTTTGACCTAAGTTTGCACGGAC
Upstream primer:
OSP-SP：CAATAGTCTCCACACCCCCCCACTATC
2. PCR the results are shown in Figure 6.
3. Fig. 7 is shown in order-checking splicing result (5423bp).
4, whole sequencing result: whole joining region sequences (joining region of the paddy rice flanking sequence of exogenous insertion vector (containing SCK gene) and both sides) is for shown in SEQ ID No.2.
5, the integrated structure of long segment checking acquisition
Increase according to 1., 2., 3., 4., 5. representing the Position Design long segment primer illustrated respectively in Fig. 7, expection size is respectively: 1.4Kb, 1176bp, 817bp, 2.3kb, 4443bp.Amplification is shown in Fig. 8, Fig. 9, Figure 10, Figure 11, Figure 12 respectively.
The screening of strain specificity qualitative detection Auele Specific Primer of rich No. 2 of experimental example 1 transgenic paddy rice section and the optimization of amplification condition
One, the screening of 5 ' end detection specificity qualitative detection Auele Specific Primer
1, the design of primer and screening
5 pairs of primers in 5 ' side wing sequences Design, primer sequence is as follows:
Primer a: amplified production clip size is 202bp
F：GATTAACCATACTGGAAAATCTGGC
R：GTCCGCAATGTGTTATTAAGTTGTC
Primer b: amplified production clip size is 156bp
F：GATTAACCATACTGGAAAATCTGGC
R：CGCAGGATAGCTAGTAGGATCG
Primer c: amplified production clip size is 142bp
F：CTTCCTGGATTTACAGCCTAAAGAT
R：GTCCGCAATGTGTTATTAAGTTGTC
Primer d: amplified production clip size is 170bp
F：ACATCGTCAGTACCATAGTGATTGC
R：GTCCGCAATGTGTTATTAAGTTGTC
Primer e: amplified production clip size is 197bp
F：CTTCCTGGATTTACAGCCTAAAGAT
R：TACTAAAATCCAGATCCCCCGA
Apply general PCR reaction conditions (primer final concentration be 0.2 μM, annealing temperature be 56 DEG C), carry out pcr amplification with above-mentioned 5 pairs of primers, 2 repetitions are set; The amplification of above-mentioned 5 pairs of primers is shown in Figure 13; From amplification, the amplification efficiency of primer a will be got well compared with the amplification efficiency of other four pairs of primers, therefore selects amplification efficiency good primer a(202bp) next step specificity verification is carried out as candidate drugs.
2, specificity verification
Checking Material selec-tion: using transgenic paddy rice section rich No. 2 as positive control, utilize the general PCR reaction conditions of application (primer final concentration be 0.2 μM, annealing temperature be 56 DEG C) to detect following 30 kinds
Paddy rice: bright extensive 63, Anhui 21-B, middlely spend 11, excellent bright extensive 86, extensive No. 1 of the China of Shanyou 63, rich No. 6 of section, rich No. 6 of the excellent section of II, II, Shan are excellent 10, bar68-1, Anhui 80-4B, anti-excellent 97, eastern agriculture, Liaojing 371, loose round-grained rice 6;
Corn: feminine gender, MON863, Bt176, MON810;
Cotton: feminine gender, LL25, MON15985;
Soybean: feminine gender, MON89788,40-3-2;
Rape: GT73(has CTPI gene), T45, MS1;
Beet: H7-1;
Detected result is shown in Figure 14.From detected result, this to primer except section rich No. 2 have amplified band except, extensive No. 1 in China, anti-excellent 97, Liaojing 371, MON89788, H7-1 also all have amplified band, so the specificity adopting this primer to detect is bad.
Two, the screening of 3 ' end flanking sequence detection specificity primer
1, the design of primer and amplification
5 pairs of specificity of transformant primers are designed according to rich No. 23 ' the end flanking sequences of section
Primer sequence is as follows:
Primer 1: amplified production clip size is 132bp
F：TAGTGTATTGACCGATTCCTTGC（SEQ ID No.3）
R：GCACGGACTATACAAGTTGTGATGT（SEQ ID No.4）
Primer 2: amplified production clip size is 172bp
F：TAGTGTATTGACCGATTCCTTGC（SEQ ID No.5）
R：CAATGAGCTATATGTGTTACAACCG（SEQ ID No.6）
Primer 3: amplified production clip size is 201bp
F：TAGAGCAGCTTGAGCTTGGATC（SEQ ID No.7）
R：GCACGGACTATACAAGTTGTGATGT（SEQ ID No.8）
Primer 4: amplified production clip size is 192bp
F：GCATATGAAATCACACCATGTAGTG（SEQ ID No.9）
R：CAATGAGCTATATGTGTTACAACCG（SEQ ID No.10）
Primer 5: amplified production clip size is 152bp
F：GCATATGAAATCACACCATGTAGTG（SEQ ID No.11）
R：GCACGGACTATACAAGTTGTGATGT（SEQ ID No.12）
The joining region sequence of amplification exogenous insertion vector and rice genome, the size of amplified production is all not less than 100bp.Apply general PCR reaction conditions, carry out pcr amplification with 5 pairs of primers, 2 repetitions are set; From amplification, these 5 pairs of primers all can amplify expection fragment specifically from rich No. 2 samples of section, but with the band the most clear (Figure 15) of the 201bp of the primer amplification shown in SEQ ID No.7 and SEQ ID No.8; All can stablize in parallel at 10 when the rich No. 2 paddy rice content of transgenosis section are 0.05% with the primer shown in SEQ ID No.7 with SEQ ID No.8 and amplify expection size strip, illustrate that this reaches 0.05% to the sensitivity that primer detects; Therefore, the present invention preferably adopts primer shown in SEQ ID No.7 and SEQ ID No.8 to detect primer as the strain specificity PCR detecting rich No. 2 of transgenic paddy rice section.
2.PCR reaction system and condition optimizing
The final concentration of primer in adjusting and optimizing PCR reaction system, arrange 0.1 μM, 0.2 μM, 0.4 μM, 4 concentration gradients such as 0.8 μM of grade, and with 54 DEG C, 56 DEG C, 58 DEG C, 60 DEG C, 62 DEG C are carried out the amplification of PCR reaction for annealing temperature, result shows, the fragment of expection size can be amplified under set different primers concentration and different annealing temperature condition, but be 0.2 μM at primer final concentration, effect best (Figure 16: primer concentration is 0.1 μM/L when annealing temperature is 58 DEG C, Figure 17: primer concentration is 0.2 μM/L, Figure 18: primer concentration is 0.4 μM/L, Figure 19: primer concentration is 0.8 μM/L).
The event-specific detection system of rich No. 2 of experimental example 2 transgenic paddy rice section and the foundation of response procedures
One, the extraction of plant genome DNA and detection
1 DNA of plants is extracted
The preparation of 1.1CTAB Extraction buffer
81.7g NaCl and 20g CTAB is added in 600mL water, 1mol/LTris-HCl(pH7.5 is added after abundant dissolving) solution 100mL, 0.5mol/L EDTA(pH8.0) solution 100mL, finally adjusts pH to 8.0 by HCl or NaOH solution, adds water and be settled to 1000mL.Use after sterilizing 20min under High Temperature High Pressure (103.4kPa/121 DEG C) condition.
1.2 extracting method
A.100mg sample, fully pulverizing lastly is transferred in 2ml centrifuge tube;
B.1ml be preheated to the CTAB Extraction buffer of 65 DEG C, fully mixing, suspension sample, and softly mix.C.65 DEG C water-bath 40min, period puts upside down mixing for several times;
D.12000r/min centrifugal 15min.The new centrifuge tube of transfer supernatant to, adds equal-volume phenol, chloroform-isoamyl alcohol (24:1), fully mixes;
E.12000r/min centrifugal 10min.The new centrifuge tube of transfer supernatant to, adds equal-volume chloroform-isoamyl alcohol (24:1), fully mixes;
F.12000r/min centrifugal 10min, gets supernatant, adds 2/3 volume isopropanol, 1/10 volumes of acetic acid sodium.Place 2 hours or the longer time for-20 DEG C;
G.12000r/min centrifugal 10min;
H. abandon supernatant, add 500 μ L, 70% ethanolic soln, and put upside down centrifuge tube for several times.The centrifugal 10min of 12000r/min;
I. abandon supernatant, centrifuge tube is stood upside down in thieving paper and blot, and ethanol is fully volatilized, dry DNA.
G. 100 μ l water dissolution DNA are added;
K. be 100ng/ μ l with double distilled water by DNA solution Concentration Modulation, be stored in-20 DEG C for subsequent use.
2DNA detects
Get the DNA solution that 3ul extracts, with the agarose gel electrophoresis of 0.8%, judge the quality of DNA according to its brightness and banding pattern.Adopt ultraviolet spectrophotometer to measure put forward concentration and the purity of DNA.
Two, the foundation of the rich No. 2 rice strain specificity qualitative PCR detection system of section and response procedures
Through optimizing, the system that the rich No. 2 rice strain specificity qualitative PCRs of section detect is in table 1, and PCR response procedures is in table 2.
The rich No. 2 rice strain specificity qualitative PCR detection system of table 1 section
The rich No. 2 rice strain specificity qualitative PCR response procedures of table 2 section
The specificity analyses of the rich No. 2 rice strain specificity qualitative PCR detection methods of experimental example 3 section
One, experiment material
1, vegetable material paddy rice
Transgenic paddy rice: excellent bright extensive 86, rich No. 6 of the excellent section of Π of Bar68-1, Π, Shan are excellent 10, Shanyou 63, Anhui 80-4B, anti-excellent 97, extensive No. 1 of China, Anhui 21-B, rich No. 6 of section, eastern agriculture etc.
Conventional rice: brightly extensively 63, middle spend 11, Liaojing 371, loose round-grained rice 6;
Other transgenic plant: corn: feminine gender, MON863, Bt176, MON810;
Cotton: feminine gender, LLcotton25, MON15985;
Soybean: feminine gender, MON89788,40-3-2;
Rape: GT73(has CTPI gene), T45, MS1;
Beet: H7-1;
Above experiment material provided by Ministry of Agriculture's development in science and technology center and the Chinese Academy of Agricultural Sciences's biology.
Two, experimental technique and result
With SEQ ID No.7 and SEQ ID No.8 for Auele Specific Primer pair, the rich No. 2 rice strain specificity qualitative PCR detection methods of the section set up according to experimental example 2 carry out amplification to various material and show, in rich No. 2 rice genomes of section, amplification obtains the band (201bp) of object clip size, rich No. 6 of the excellent section of other transgenic paddy rices Π, Shan excellent 10, Shanyou 63, Anhui 80-4B, anti-excellent 97, China extensive No. 1 and other transgenic plant MON863 corns, GHB614 cotton, MON89788 soybean and modified rape GT 73, and do not have in the genome such as conventional rice bright extensive 63 to increase to obtain fragment (Figure 20 of object clip size, Figure 21).Consistent with object amplified fragments sequence by sequencing analysis show to increase in rich No. 2 rice genomes of the section sequence that obtains.The above results shows that the rich No. 2 strain specificity qualitative PCR detection methods of transgenic paddy rice section that primer Kefeng2-F and Kefeng2-R sets up have high degree of specificity.
The sensitivity experiment of the rich No. 2 rice strain specificity qualitative PCR detection methods of experimental example 4 section
With SEQ ID No.7 and SEQ ID No.8 for Auele Specific Primer pair, the rich No. 2 rice strain specificity qualitative PCR detection methods of the section set up according to experimental example 3 carry out sensitive amplification experiment.
The rich No. 2 positive DNA of section and bright extensive 86 negative DNA press different mass than mixing, rich No. 2 massfractions of preparation section are respectively the sample of 1%, 0.5%, 0.1%, 0.05%, 0.01%, the reaction system of testing according to specificity and response procedures carry out pcr amplification, experimental result shows, the rich No. 2 paddy rice compositions of transgenic paddy rice section are 1%, 0.5%, 0.1%, all amplified the object fragment that size is 201bp in the PCR reaction system of 0.05%, the sensitivity of the rich No. 2 rice strain specificity qualitative PCR detection methods of explanation section can reach 0.05%(Figure 22).
The detectability experiment of the rich No. 2 rice strain specificity qualitative PCR detection methods of experimental example 5 section
The rich No. 2 positive DNA of section and bright extensive 86 negative DNA press different mass than mixing, rich No. 2 massfractions of preparation section are respectively 0.1%, 0.05%, the sample of 0.01%, the reaction system of testing according to specificity and response procedures carry out pcr amplification, by experimental result visible (Figure 23), when the content of rich No. 2 paddy rice of section is 0.1% and 0.05%, all can stablize in parallel at 10 and amplify expection size strip, and this band presents unstable in 10 parallel middle amplifications of the positive 0.01%, therefore the detection of the rich No. 2 rice strain specificity qualitative PCR detection methods of section of the present invention is limited to 0.05%.
Claims (2)
1. the strain specificity PCR for detecting rich No. 2 of transgenic paddy rice section detects primer, it is characterized in that, is selected from any pair in following 4 pairs of primers: nucleotides sequence is classified as the primer pair 1 shown in SEQ ID No.3 and SEQ ID No.4; Nucleotides sequence is classified as the primer pair 2 shown in SEQID No.5 and SEQ ID No.6; Nucleotides sequence is classified as the primer pair 4 shown in SEQ IDNo.9 and SEQ ID No.10; Nucleotides sequence is classified as the primer pair 5 shown in SEQ ID No.11 and SEQ ID No.12.
2. a strain specificity PCR qualitative detection test kit for rich No. 2 of transgenic paddy rice section, comprising: 10 × PCR damping fluid, magnesium chloride solution, dNTPs mixing solutions, detects upstream and downstream primer pair, Taq enzyme and distilled water; It is characterized in that: described detection upstream and downstream primer is that PCR according to claim 1 detects primer.
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