CN107475391A - Arctic apple specificity real-time fluorescent PCR testing primer, probe, method and kit - Google Patents
Arctic apple specificity real-time fluorescent PCR testing primer, probe, method and kit Download PDFInfo
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Abstract
The invention discloses arctic apple specificity real-time fluorescent PCR testing primer, probe, method and kit.The present invention is directed to arctic apple specificity PGAS NOS adjacent area primers and probe, establish arctic apple specificity real-time fluorescence PCR detection method, there is good specificity, high sensitivity, repeatable good, amplification efficiency 96%, minimum quantitative Monitoring lower-cut is 20copies.It is effective to solve the problems, such as in the prior art without the accurate quantitative measurement technology of arctic apple specificity, this method can be applied to pass in and out Check and Examination of Port quarantine, domestic agricultural products foods supervision arctic apple and products thereof detection.
Description
Technical field：
The invention belongs to GMO detection field, and in particular to arctic apple specificity real-time PCR detection draws
Thing, probe, method and kit.
Background technology：
Arctic apple is that (Okanagan Specialty Fruits, are called in the following text by Canadian oka Nuo Gen characteristic fruits company
Oka Nuo Gen companies) research and development BPH resistant rice variety kind, brand name is arctic apple (ArcticTM) at present approval listing arctic apple
Strain shares three：The Arctic of America ＆ Canada approval listing in 2015TM" Golden Delicious " (GD743) and
ArcticTMGranny (GS784), the Arctic of U.S.'s approval listing in 2016TMFuji(NF872)。
Arctic apple (ArcticTM) series by agriculture bacillus mediated carrier GEN-03 insert respectively gold hat (Golden
Dilicious, GD743), green apple (Granny Smith, GS784) and Fuji (Fuji, NF872) develop, GEN-03
Carrier includes chimeric PPO (polyphenol oxidase) and suppresses sequence, four genes of the gene families of PPO containing apple (PPO2, GPO3,
APO5 and pSR7, abbreviation PGAS) positive sequence, they are opened by CaMV 35S (Cauliflower Mosaic Virus 35S)
Mover and the regulation and control of NOS (nopaline synthase) terminator.Arctic apple (ArcticTM) main foreign gene is PPO suppressions
Gene and nptII processed.Arctic apple (ArcticTM) it is the expression that whole PPO gene families in apple are reduced using RNAi technology
And produce the apple strain of BPH resistant rice variety.Wherein PPO suppressors produce double-stranded RNA (double stranded RNA, dsRNA),
The process that it suppresses transcription is complementary directly by sequence by siRNA (small interfering RNAs, siRNAs)
Shearing target mRNA produces BPH resistant rice variety apple so as to suppress PPO expression.Wherein Arctic Fuji are in the 3rd, 13 and No. 17 dyeing
There are multiple GEN-03 fragments to insert on body, sequence PPO may be that 4 functionals copy, Arctic Golden insertion
PPO may be 1-2 copy, and Arctic Granny may be 4 copies.
At present to transgenic product majority state using corresponding mark management system, identify management system foundation and
Implement dependent on effectively accurate Testing and appraisal technology.To break the transgenic product Trade technique of other countries and area setting
Barrier, improve and China's transgenic product quantitative measurement technology system, right to know and choosing of the protection consumer to transgenic product
The power of selecting, the method for providing arctic apple Testing and appraisal for the port supervision department that passes in and out, it is special to establish structure for arctic apple
Property real time PCR detection method.The other countries such as European Union and area there is no arctic apple specificity real-time PCR detection at present
Method.
The content of the invention：
It is an object of the invention to provide a species specificity is good, high sensitivity, stability are strong, quick and precisely differentiate arctic apple
(including tri- strains of GD743, GS784 and NF872) specific real-time fluorescent PCR testing primer, probe, method and kit.
First purpose of the present invention is to provide a kind of arctic apple specificity real-time fluorescent PCR testing primer, its feature
It is, described detection primer is as follows：
Arctic-F：5 '-TGACCCGAACCAGACCCAC-3 ' (as shown in SEQ ID NO.1)；
Arctic-R：5 '-CAGGATTCAATCTTAAGAAACTTTATTG-3 ' (as shown in SEQ ID NO.2).
Second object of the present invention is to provide a kind of arctic apple specificity real-time PCR detection probe, its feature
It is, described detection probe is as follows：
Arctic-P：5 '-TAACTTCTACTCCGCGGCTCGATCG-3 ' (as shown in SEQ ID NO.3), the 5 ' of probe
End is marked with fluorescent reporter group, and 3' ends are marked with fluorescent quenching group.
Described fluorescent reporter group is preferably FAM, and described fluorescent quenching group is preferably BHQ1.
Third object of the present invention is to provide a kind of arctic apple specificity real-time fluorescence PCR assay kit, including
Real-time fluorescence quantitative PCR reaction solution, hot resistant DNA polymerase, detection primer and detection probe, it is characterised in that described detection
Primer is as follows：
Arctic-F：5 '-TGACCCGAACCAGACCCAC-3 ' (as shown in SEQ ID NO.1)；
Arctic-R：5 '-CAGGATTCAATCTTAAGAAACTTTATTG-3 ' (as shown in SEQ ID NO.2)；
Described detection probe is as follows：
Arctic-P：5 '-TAACTTCTACTCCGCGGCTCGATCG-3 ' (as shown in SEQ ID NO.3), the 5 ' of probe
End is marked with fluorescent reporter group, and 3' ends are marked with fluorescent quenching group.
Fourth object of the present invention is to provide a kind of arctic apple specificity real-time fluorescence PCR detection method, its feature
It is, comprises the following steps：
(1) genomic DNA of sample is extracted as template；
(2) above-mentioned detection primer and detection probe are added, is polymerize with real-time fluorescence quantitative PCR reaction solution and heat-resistant dna
Enzyme is mixed to form amplification reaction system；
(3) amplification reaction system is subjected to real-time fluorescence PCR reaction on fluorescence PCP instrument, after reaction terminates, according to amplification
Whether curve judgement sample is arctic apple.
It is preferred that the amplification reaction system of described step (2) is：25 μ L, including Premix Ex Taq 12.5 μ L, ROX
Reference Dye II 0.2 μ L, 10 μm of each 0.5 μ L of ol/L detection primers Arctic-F and Arctic-R, 10 μm of ol/L detections
The μ L of probe Arctic-P 0.5, DNA profiling 2 μ L and ddH2O 8.8μL；The real-time fluorescence PCR reaction of described step (3), its
Response procedures are 95 DEG C of 30s；95 DEG C of 5s, 60 DEG C of 34s, 40 circulations, fluorescence signal is collected in 60 DEG C.
It is preferred that whether described step (3) is that the standard of arctic apple is according to amplification curve judgement sample：If expand
Increasing curve has Representative fluorescence amplification curve, then it is arctic apple to illustrate sample；If amplification curve without Representative fluorescence amplification curve,
It is not arctic apple then to illustrate sample.
The 5th purpose of the present invention is to provide a kind of arctic apple specificity real-time fluorescent PCR quantitative detection method, its
It is characterised by, comprises the following steps：
(1) genomic DNA for extracting arctic apple carries out template of the gradient dilution for use as different initial concentrations, adds respectively
Enter above-mentioned detection primer and detection probe, amplification is mixed to form with real-time fluorescence quantitative PCR reaction solution and hot resistant DNA polymerase
Reaction system, real-time fluorescence PCR reaction is carried out on fluorescent PCR instrument；
(2) Ct values corresponding to each initial concentration template are obtained after reacting, the common logarithm of the Ct values and starting template amount
(lg) it is linear, the standard curve of arctic apple in the range of linearity is obtained accordingly；
(3) genomic DNA for extracting testing sample is template, adds the above-mentioned detection with same system in step (1)
Primer and detection probe, amplification reaction system is mixed to form with real-time fluorescence quantitative PCR reaction solution and hot resistant DNA polymerase,
Real-time fluorescence PCR reaction is carried out on fluorescence PCP instrument, Ct values are obtained after reaction, is substituted into the arctic apple of step (2) acquisition
Calibration curve equation, the content (copy number or quality) of apple genome DNA in the arctic in measuring samples is calculated.
The present invention for tri- strains of the arctic apple GD743, GS784 and NF872 share specific sequence (PGAS and
NOS abuts region sequence) primer and TaqMan probe are designed, establish arctic apple real-time fluorescent polyase chain reaction
(polymerase chain reaction, PCR) detection method, using the detection primer and detection probe, according to the present invention's
Method carries out detection and finds that its quantitative Monitoring lower-cut is 20 copies, and the standard curve linearly dependent coefficient (R2) of foundation is 0.99,
Amplification efficiency E is 96%, and repeated experiment shows that the standard deviation (SD) of this method and relative standard deviation (RSD) can all connect
By in the range of, specific good, high sensitivity, stability are strong, can meet that supervision department and the public carry out strain to arctic apple
The demand of specific detection identification.
Brief description of the drawings：
Fig. 1 is apple composition specific amplification figure, and 1~7 is respectively Fuji apple, yellow golden marshall apple, green apple (Granny
Smith), queen's apple (Queen), red rose apple (Pacfic Rose), jazz's apple (Jazz), Gala apple (Gala)
Amplification curve, feminine gender amplification are respectively：Pears, mangosteen (Garcinia mangostana L.), nectarine (Nectarine), lichee
(Litchi chinensis Sonn.), longan (Dimocarpus longan), transgenic corns MIR162, beet H7-1, the moon
Property control and blank control.
Fig. 2 is arctic apple method for detecting specificity specificity experiments result, and 1 is Malus-Arctic plasmids；2~14 points
It is not：Fuji apple, yellow golden marshall apple, green apple (Granny Smith), queen's apple (Queen), red rose apple
(Pacfic Rose), jazz's apple (Jazz), Gala apple (Gala), pears, mangosteen (Garcinia mangostana L.),
Nectarine (Nectarine), lichee (Litchi chinensis Sonn.), longan (Dimocarpus longan), transgenosis are beautiful
Rice MIR162, beet H7-1, negative control and blank control.
Fig. 3 is arctic apple specific detection sensitivity test amplification figure；1~7 is respectively from left to right：1000000、
100000th, 10000,1000,100,10 and 5copies/ μ L DNAs.
Fig. 4 is the standard curve of real-time PCR detection arctic apple, and Quantity represents DNA profiling amount (copy number).
Embodiment：
Following examples are to further explanation of the invention, rather than limitation of the present invention.
Main material：It is Fuji apple, yellow golden marshall apple, green apple (Granny Smith), queen's apple (Queen), red
Rose apple (Pacfic Rose), jazz's apple (Jazz), Gala apple (Gala), pears, mangosteen (Garcinia
Mangostana L.), nectarine (Nectarine), lichee (Litchi chinensis Sonn.), longan (Dimocarpus
Longan), transgenic corns MIR162, beet H7-1 are that deposit, apple endogenous gene apple ribosomes are purchased in this laboratory
(dual-gene positive plasmid is will be as by ITS1-5.8S and the dual-gene positive plasmid Malus-Arctic of arctic apple specific fragment
Fragment shown in SEQ ID NO.6 is cloned into the recombinant plasmid for building to obtain on the PUC57 carriers of AmpR resistances, by recombinant plasmid
It is transferred to -70 DEG C of preservations after recipient bacterium DH5a.Hereinafter referred to as Malus-Arctic plasmids.Wherein, the fragment as shown in SEQ ID NO.6
Including endogenous gene ITS1-5.8S genetic fragment 100bp sequences (as shown in SEQ ID NO.4), arctic apple specific fragment
BamHI restriction enzyme site (ggatcc) of the 277bp sequences (as shown in SEQ ID NO.5) between two fragments) it is this laboratory
Structure.
Select apple ITS1-5.8S ribosomal RNA genes (Malus asiatica internal transcribed
Spacer 1,5.8S ribosomal RNA gene, GenBank:AF186494.1) primer Malus-F/R and probe Malus-
P is used to detect whether apple sample gene group DNA successfully extracts and whether be adapted for real-time fluorescent PCR amplification；Its sequence
Information refers to table 1.
Main agents：Primex Ex Taq (2 ×) for qPCR, the precious biology in Dalian；DNA extraction kit, Beijing Tiangeng
Company；Primer and probe is synthesized by Shan Jing biotech firms, is diluted to final concentration of 10 μM of working solution and is used.
Key instrument and equipment：
ABI7500, ABI7500FAST real-time fluorescence quantitative PCR instrument, Applied biosystems；The droplets of QX 200
Formula digital pcr system, Bio Rad Laboratories；Nanodrop2000c micro-spectrophotometers, Thermo companies of the U.S.；Grinder,
German IKA.
Crop material sample gene group DNA uses conventional method extraction and purification.
Embodiment 1：The foundation of real-time fluorescence PCR detection method
According to structural specificity sequence (as shown in SEQ ID NO.5) between the arctic apple PGAS and NOS, using Primer
The software Design primers of Primer 5.0 and probe.Apple ITS1-5.8S ribosomal RNA genes are for apple source sample DNA's
Detection and the relative quantification of transgene component.Specific primed probe information is shown in Table 1.
Primer, the probe of the real-time fluorescence PCR of table 1
Amplification reaction system is 25 μ L：Including Premix Ex TaqTM12.5 μ L, ROX Reference Dye II 0.2
μ L, 10 μm of ol/L sense primers and anti-sense primer each 0.5 μ L, 10 μm of μ L of ol/L detection probes 0.5, DNA profiling 2 μ L and ddH2O
8.8μL；Response procedures are 95 DEG C of 30s；95 DEG C of 5s, 60 DEG C of 34s, 40 circulations, fluorescence signal is collected in 60 DEG C.
Embodiment 2：Real time fluorescent PCR method specific test
Using Fuji apple, yellow golden marshall apple, green apple (Granny Smith), queen's apple (Queen), red rose apple
Fruit (Pacfic Rose), jazz's apple (Jazz), Gala apple (Gala), pears, mangosteen (Garcinia mangostana
L.), nectarine (Nectarine), lichee (Litchi chinensis Sonn.), longan (Dimocarpus longan), turn base
Because corn MIR162, beet H7-1 genomic DNAs are template.To the special of apple endogenous gene real-time fluorescence PCR detection method
Property is tested.Real-time fluorescence PCR detecting reaction system and response procedures are the same as embodiment 1.
As a result show, the DNA of the sample of all extractions is carried out using primer Malus-F/R and probe Malus-P real-time
During fluorescent PCR, only Fuji apple, yellow golden marshall apple, green apple (Granny Smith), queen's apple (Queen), red rose
There is amplification curve in the DNA profiling of apple (Pacfic Rose), jazz's apple (Jazz) and Gala apple (Gala) source, its
His crops show the correct (figure of apple endogenous gene ITS1-5.8 ribosomal RNA genes amplification without typical amplification curve
1)。
Using Fuji apple, yellow golden marshall apple, green apple (Granny Smith), queen's apple (Queen), red rose apple
Fruit (Pacfic Rose), jazz's apple (Jazz), Gala apple (Gala), pears, mangosteen (Garcinia mangostana
L.), nectarine (Nectarine), lichee (Litchi chinensis Sonn.), longan (Dimocarpus longan), turn base
Because corn MIR162, beet H7-1 genomic DNAs are template, positive is Malus-Arctic plasmids, and negative control is non-
Genetically modified rice DNA.The specificity of the arctic apple real-time fluorescence PCR detection method of foundation is tested.Real-time fluorescence PCR
Reaction system and response procedures are detected with embodiment 1.
The DNA of the sample of all extractions is entered using the arctic apple specific primer Arctic-F/R and probe Arctic-P
During row real-time fluorescence PCR, the DNA profiling of only positive Malus-Arctic plasmids has Representative fluorescence amplification curve, with it
His crop material DNA is template without Representative fluorescence amplification curve (Fig. 2).Show that the detection method specificity of the present invention is good
It is good.
Embodiment 3：Sensitivity test, repeatability test and standard curve are established
Add TE buffer solutions to be diluted to 1000000 respectively the Malus-Arctic plasmid DNA solutions of extraction, 100000,
10000th, 1000,100,10 and 5copies/ μ L carry out arctic apple real-time PCR detection, carried out linear as DNA profiling
Range test and repeatability test, each sample carry out 9 repetitions and tested, and water is blank control, and real-time PCR detection is anti-
System and response procedures are answered with embodiment 1.Test result shows (table 2 and Fig. 3), the 1-7 in the range of 2000000-10copies
7 concentration gradients 9 it is parallel can have typical amplification curve, so minimal detectable concentration (LOD) is 10copies (Fig. 3)；
In table 2, the SD and RSD of Ct values are slightly higher obtained by 10copies concentration, more unstable than 20copies acquired results, therefore test
LOQ is ideal in 20copies；In addition, in table 2 SD of each 9 parallel gained Ct values of 7 concentration between 0.04~0.35,
RSD shows that repeatability is good between 0.18~0.96, respectively less than 25%.7 in the range of template amount 10-2000000copies
The logarithm value of concentration establishes standard curve (Fig. 4) with gained Ct values, and equation of linear regression is：Y=-3.43x+39.579, R2 are
0.998, amplification efficiency is 96% (between 90%~110%), shows that its linear dependence is good, and amplification efficiency is high, meets
ENGL related requests [Definition of Minimum Performance Requirements for Analytical
Methods of GMO Testing]。
The sensitivity and repeatability test of the real time fluorescent PCR method of table 2
The present invention is special for arctic apple PGAS and NOS adjacent areas primers and probe, the arctic apple of foundation
Different in nature real-time fluorescence PCR detection method, quick, the high-throughout qualitative and quantitative detection of specificity can be carried out to arctic apple, it is full
The demand that foot detection, supervision department are identified to it.
Sequence table
<110>Complex art service centre of Huangpu Entry-Exit Inspection and Quarantine Bureau
<120>Arctic apple specificity real-time fluorescent PCR testing primer, probe, method and kit
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
tgacccgaac cagacccac 19
<210> 2
<211> 28
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
caggattcaa tcttaagaaa ctttattg 28
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
taacttctac tccgcggctc gatcg 25
<210> 4
<211> 100
<212> DNA
<213>Apple (Malus pumila Mill)
<400> 4
ggaatctgaa cgaaagagcg cgctcccgcc gccccggaaa cggtgcgcgc gcgggtgcgt 60
cgtcgtcttc gataagtcaa aacgactctc ggcaacggat 100
<210> 5
<211> 277
<212> DNA
<213>Arctic apple (Arctic Apples)
<400> 5
ctacgccggc accattgaga acagtcctca taataacatc catctctggt gcggtgaccc 60
gaaccagacc caccacgaag acatgggtaa cttctactcc gcggctcgat cgttcaaaca 120
tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg attatcatat 180
aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg acgttattta 240
tgagatgggt ttttatgatt agagtcccgc aattata 277
<210> 6
<211> 383
<212> DNA
<213>Malus-Arctic plasmids (Malus-Arctic plasmid)
<400> 6
ggaatctgaa cgaaagagcg cgctcccgcc gccccggaaa cggtgcgcgc gcgggtgcgt 60
cgtcgtcttc gataagtcaa aacgactctc ggcaacggat ggatccctac gccggcacca 120
ttgagaacag tcctcataat aacatccatc tctggtgcgg tgacccgaac cagacccacc 180
acgaagacat gggtaacttc tactccgcgg ctcgatcgtt caaacatttg gcaataaagt 240
ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat 300
tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt 360
atgattagag tcccgcaatt ata 383
Claims (8)
- A kind of 1. arctic apple specificity real-time fluorescent PCR testing primer, it is characterised in that the following institute of described detection primer Show：Arctic-F：5’-TGACCCGAACCAGACCCAC-3’；Arctic-R：5’-CAGGATTCAATCTTAAGAAACTTTATTG-3’.
- A kind of 2. arctic apple specificity real-time PCR detection probe, it is characterised in that the following institute of described detection probe Show：Arctic-P：5 '-TAACTTCTACTCCGCGGCTCGATCG-3 ', 5 ' ends of probe are marked with fluorescent reporter group, 3' End is marked with fluorescent quenching group.
- 3. arctic apple specificity real-time PCR detection probe according to claim 2, it is characterised in that described Fluorescent reporter group is FAM, and described fluorescent quenching group is BHQ1.
- 4. a kind of arctic apple specificity real-time fluorescence PCR assay kit, including it is real-time fluorescence quantitative PCR reaction solution, heat-resisting Archaeal dna polymerase, detection primer and detection probe, it is characterised in that described detection primer is as follows：Arctic-F：5’-TGACCCGAACCAGACCCAC-3’；Arctic-R：5’-CAGGATTCAATCTTAAGAAACTTTATTG-3’；Described detection probe is as follows：Arctic-P：5 '-TAACTTCTACTCCGCGGCTCGATCG-3 ', 5 ' ends of probe are marked with fluorescent reporter group, 3' End is marked with fluorescent quenching group.
- 5. a kind of arctic apple specificity real-time fluorescence PCR detection method, it is characterised in that comprise the following steps：(1) genomic DNA of sample is extracted as template；(2) detection probe described in the detection primer and claim 2 described in claim 1 is added, with real-time fluorescence quantitative PCR Reaction solution and hot resistant DNA polymerase are mixed to form amplification reaction system；(3) amplification reaction system is subjected to real-time fluorescence PCR reaction on fluorescence PCP instrument, after reaction terminates, according to amplification curve Whether judgement sample is arctic apple.
- 6. apple specificity real-time fluorescence PCR detection method in the arctic according to claim 5, it is characterised in that described The amplification reaction system of step (2) is：25 μ L, including μ L, the ROX Reference Dye II of Premix Ex Taq 12.5 0.2 μ L, 10 μm of ol/L detection primers Arctic-F and Arctic-R each 0.5 μ L, 10 μm of μ of ol/L detection probes Arctic-P 0.5 L, DNA profiling 2 μ L and ddH2O 8.8μL；The real-time fluorescence PCR reaction of described step (3), its response procedures is 95 DEG C of 30s； 95 DEG C of 5s, 60 DEG C of 34s, 40 circulations, fluorescence signal is collected in 60 DEG C.
- 7. apple specificity real-time fluorescence PCR detection method in the arctic according to claim 5, it is characterised in that described Whether step (3) is that the standard of arctic apple is according to amplification curve judgement sample：If amplification curve has Representative fluorescence expansion Increase curve, then it is arctic apple to illustrate sample；If for amplification curve without Representative fluorescence amplification curve, it is not the arctic to illustrate sample Apple.
- 8. a kind of arctic apple specificity real-time fluorescent PCR quantitative detection method, it is characterised in that comprise the following steps：(1) genomic DNA for extracting arctic apple carries out gradient dilution for use as the template of different initial concentrations, is separately added into power Profit requires the detection probe described in detection primer and claim 2 described in 1, with real-time fluorescence quantitative PCR reaction solution and heat-resisting Archaeal dna polymerase is mixed to form amplification reaction system, and real-time fluorescence PCR reaction is carried out on fluorescent PCR instrument；(2) Ct values corresponding to each initial concentration template are obtained after reacting, the common logarithm (lg) of the Ct values and starting template amount is in Linear relationship, the standard curve of arctic apple in the range of linearity is obtained accordingly；(3) genomic DNA for extracting testing sample is template, in addition and step (1) described in the claim 1 of same system Detection probe described in detection primer and claim 2, mixed with real-time fluorescence quantitative PCR reaction solution and hot resistant DNA polymerase Amplification reaction system is formed, real-time fluorescence PCR reaction is carried out on fluorescence PCP instrument, Ct values are obtained after reaction, are substituted into step (2) calibration curve equation of the arctic apple obtained, the content of apple genome DNA in the arctic in measuring samples is calculated.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112126699A (en) * | 2020-09-15 | 2020-12-25 | 中国农业大学 | Malus plant complete genome InDel marker genotype database and application thereof in germplasm resource specificity identification |
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| CN105154573A (en) * | 2015-10-21 | 2015-12-16 | 河北科技师范学院 | Primer set and probe for apple Actin gene real-time fluorescent quantitative PCR (polymerase chain reaction) detection and detection method by using same |
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| CN101392296A (en) * | 2008-11-07 | 2009-03-25 | 中国农业大学 | Apple S gene type rapid identification method and special kit thereof |
| WO2011098907A2 (en) * | 2010-02-15 | 2011-08-18 | Council Of Scientific & Industrial Research | Method for detecting fungal pathogens |
| CN102344952A (en) * | 2010-07-30 | 2012-02-08 | 中国检验检疫科学研究院 | Primer, method and kit for detecting apple-derived materials in sample |
| CN105154573A (en) * | 2015-10-21 | 2015-12-16 | 河北科技师范学院 | Primer set and probe for apple Actin gene real-time fluorescent quantitative PCR (polymerase chain reaction) detection and detection method by using same |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112126699A (en) * | 2020-09-15 | 2020-12-25 | 中国农业大学 | Malus plant complete genome InDel marker genotype database and application thereof in germplasm resource specificity identification |
| CN112126699B (en) * | 2020-09-15 | 2022-03-01 | 中国农业大学 | Malus plant complete genome InDel marker genotype database and application thereof in germplasm resource specificity identification |
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