CN103421893B - The LAMP detection reagent of transgenic papaya GM YK strain - Google Patents
The LAMP detection reagent of transgenic papaya GM YK strain Download PDFInfo
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Abstract
Do you the invention discloses transgenic papaya GM? the LAMP detection reagent of YK strain, in addition primers F 3 and B3, inner primer FIP and BIP, ring primer Loop1 and Loop2 amplified target sequence, have special, responsive advantage; The key of LAMP technology is 6 ~ 8 zone design, the three pairs of special primers for target gene, do you establish antiviral transgenic papaya GM? YK event-specific detection reagent, by fluorescence dye SYBR? Green? I process amplified production, realize observations visual, the requirement that the transgenic papaya whether inspection transgenic papaya reaches inspection and quarantining for import/export and whether meet China is produced.Simple to operate during detection, only need a simple thermostat water bath, with low cost, be easy to operate at the scene or basic unit applies.
Description
Technical field
The present invention relates to the reagent detecting papaya, be specifically related to the LAMP detection reagent of transgenic papaya GMYK strain.
Background technology
Papaya (Caricapapaya), also known as pawpaw, is cruciate flower order Caricaceae fruit.The U.S. was devoted to the transgenic papaya research of developing anti-PRSV from 1985, within 1989, carried out Cloning and sequencing to the CP gene of PRSV strain HA5-1, started the via Particle Bombardment Transformation of papaya embryo.[the FitchMMM such as Fitch, ManshardtRM, GonsalvesD, etal.Stabletransformationofpapayaviamicroprojectilebomba rdment.PlantCellRep, 1990,9:189 ~ 194] PRSV capsid protein (Coatprotein, CP) gene of successfully encoding turns people's papaya, obtain the disease-resistant strain " 55-1 " of Transgenic cp, and got permission to enter merchandized handling in 1997.Li Hua equality [RuanXL, LiHP, ZhouGH.EvaluationofPRSVresistanceofT2transgenicpapayawit hreplicasegene.JournalofSouthChinaAgriculturalUniversity, 2004,4:12-15] rdrp gene (replicase, RP) of PRSVYS is proceeded to main breed after obtain, in China mainland, there is resistance transgenic strain " No. 1, magnificent agriculture ".The CP gene transformation of PRSVYK strain in native breed, is obtained the transgenosis pawpaw strain " GMYK " other PRSV strains (Hawaii, Thailand and Mexico) to resistance of wide spectrum by Taiwan Cheng etc.[the Cheng such as Taiwan Chung Hsing University Ye Xidong, Yeh.InvivoexpressionofPRSVCPgeneswithdifferentleadersBot [J] .BullAcadSin, 2000,41:1-10] adopt the liquid phase treatment of wounds pawpaw immature embryo method created, the CP gene of PRSVYK strain is proceeded in " No. 2, platform agriculture ", obtains transgenosis pawpaw strain " GMYK16-0-1 ", " GMYK18-2-4 "." No. 1, magnificent agriculture " is that China the 1st example is got permission to carry out commercial transgenosis fruit tree crop, within 2006, obtaining the safety certificate [Nong Jian demonstrate,prove No. 001st, word (2006)] of " No. 1, the agriculture of transgenic papaya China is in Guangdong Province's application " that the Ministry of Agriculture issues, is that uniquely a kind of approve through the Ministry of Agriculture can in the transgenic papaya strain of production and selling within Chinese territory.Transgenic papaya " GMYK " strain is the antiviral transgenic papaya with resistance of wide spectrum transformed by Taiwan Cheng etc., and the PRSV-P4 virus strain in south China area also shows higher resistance.But transgenic papaya GMYK product tie up to a lot of area of south China to be commercially produced, and sells to domestic each department in a large number.
For the various transgenic papayas that may occur in market, genetically modified crops detection method difinite quality PCR, composite PCR, real-time fluorescence quantitative PCR, test strip etc. that we commonly use, PCR in real time as the antiviral transgenic papaya of Han Jianxun etc. detects [inspection and quarantine academic periodical 2010(1) 15-20], these detection methods all have defect in various degree in efficiency, cost and operability.The reaction reagent of loop-mediated isothermal amplification technique (LAMP) comprises reaction buffer, reaction enzymes liquid, Auele Specific Primer and developer etc., this reaction reagent depends on the primer and a kind of tool de-rotation function and the BstDNA polysaccharase of the amplification in waterfall type that can identify 6 ~ 8 specific regions on target sequence, amplified target sequence that under isothermal conditions can be efficient, quick and special.Because LAMP is waterfall type amplification, therefore its amplification efficiency is very high; Meanwhile, LAMP identifies 6 ~ 8 specific position on target sequence, and its specificity is also very strong; LAMP only carries out constant-temperature amplification at 60 DEG C ~ 65 DEG C, as long as water-bath, without the need to special instrument, operates very simple; And the whole detection reaction of LAMP only needs 1.5h ~ 2.5h, sense cycle is very short.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind ofly has to transgenic papaya GMYK strain the LAMP detection reagent that specificity is high, sense cycle is short, with low cost and simple to operate.
The native gene with reference to papaya in GenBank is selected by system of the present invention, according to the boundary position of the endogenous left end of papaya and CaMV35S gene right-hand member, use PrimerExploerV4(http: //primerexlorer.jp) devise 3 group-specific primers (Pap-1, Pap-2, Pap-3), preferred through a large amount of reaction conditionss, simultaneous test and proof test, and through the detection application evaluation of a large amount of actual sample (with the DNA of transgenic papaya GMYK for template, genetically modified rice DNA is the contrast of CaMV35S positive, non-transgenic papaya DNA is negative control, water is blank, each group of primer is utilized to carry out LAMP detection, amplified production SYBRGreenI dyes.By observing the conservative property of amplified production and specificity, the suitableeest primer sets of screening), screening obtains three couples of Auele Specific Primer F3 and B3, FIP and BIP, Loop1 and Loop2 of amplification efficiency and the good Pap-3 group of specificity.Primer synthesizes by Shanghai Ying Weijie base Bioisystech Co., Ltd.
The LAMP detection reagent of transgenic papaya GMYK strain of the present invention, comprise 10 × LAMP reaction buffer, trimethyl-glycine, dNTP, BstDNA polysaccharase and three pairs of Auele Specific Primers, the nucleotide sequence of described three pairs of Auele Specific Primers is respectively:
F3：CTCATTCAGACCGGTATCC
B3：AGCAAGTGGATTGATGTGAT
FIP：CGGGGGACTCTAGAGGATCCTTTTCAGCTTCATTTTTAGACGCC
BIP：TCCTTATATAGAGGAAGGGTCTTGCTTTTCCACTGACGTAAGGGATGA
Loop1：GGGTGGTCAGTCCCTTCCAT
Loop2：GAAGGATAGTGGGATTGTGCG。
The LAMP detection reagent advantage of transgenic papaya GMYK strain of the present invention is as follows: primers F 3 and B3 in addition, inner primer FIP and BIP, ring primer Loop1 and Loop2 amplified target sequence, have special, responsive advantage; The key of LAMP technology is 6 ~ 8 zone design, the three pairs of special primers for target gene, establish antiviral transgenic papaya GMYK event-specific detection reagent, by fluorescence dye SYBRGreenI process amplified production, realize observations visual, the requirement that the transgenic papaya whether inspection transgenic papaya reaches inspection and quarantining for import/export and whether meet China is produced.Simple to operate during detection, only need a simple thermostat water bath, with low cost, be easy to operate at the scene or basic unit applies; Sense cycle is short with the naked eye just can judge LAMP result by fast qualitative, has visual result, judges the advantage that difficulty is little.We carry out detection GMOs to 10 kinds of commercially available papaya and papaya processed goods simultaneously, have detected the antiviral transgenic papaya GMYK series of Taiwan Chung Hsing University research and development.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
The LAMP detection reagent of embodiment 1, transgenic papaya GMYK strain
With 25 μ L reaction systems: the BstDNA polysaccharase 1.5 μ L of 10 × LAMP reaction buffer 2.5 μ L, concentration 8U/ μ L, concentration is the dNTP4.0 μ L of 2.5mmol/L, and concentration is the MgCl of 25mmol/L
22.5 μ L, concentration is the trimethyl-glycine 4.0 μ L of 5.0mol/L, and concentration is all each 0.2 μ L of FIP and BIP of 20 μm of ol/L, and concentration is all each 1.6 μ L of F3 and B3 of 20 μm of ol/L, and concentration is all each 0.8 μ L of Loop1 and Loop2 of 20 μm of ol/L.LAMP reaction buffer and BstDNA polysaccharase are purchased from NEWENGLANDBiolabs company, and precious biotechnology (Dalian) company limited of dNTP, trimethyl-glycine is purchased from Shanghai Chemical Reagent Co., Ltd., Sinopharm Group.
Embodiment 2, DNA extraction
Papaya DNA extraction is extracted by DneasyPlantMiniKit reagent (purchased from German QIAGEN company) operation instructions: (1) takes 200mg sample, uses pearl mill method by sample homogenization, loads in 1.5mL centrifuge tube; (2) add 500 μ L buffer A P1, vibrate 15min in the vibration constant-temperature metal bath of 65 DEG C; (3) add 130 μ L buffer A P2, turn upside down after fully mixing, in 4 DEG C of refrigerators, leave standstill 5min; (4) with the centrifugal 5min of 12000r/min room temperature, get supernatant liquor 500 μ L and go in 2mL collection tube; (5) collection tube is put into QIAcube nucleic acid automatic extracting instrument, select DNeasyPlantMini pattern, automatically extract DNA, use the biological spectrophotometric determination OD of U-0080D
260and OD
280value, calculates DNA concentration; (6) take out DNA sample template, be stored in-20 DEG C of refrigerators for subsequent use.
Embodiment 3, LAMP detect
We have purchased papaya or processed goods from supermarket, Ningbo: Hainan pawpaw, red heart pawpaw, all happy pawpaw, Thailand pawpaw, Guangxi nonage Fructus Chaenomelis, day litre melon, dry, the Taiwan nonage Fructus Chaenomelis powder of platform agriculture pawpaw, happy strange pawpaw etc.Apply LAMP of the present invention to detect, detection method comprises the DNA extraction as embodiment 2, and get each 2 μ L of DNA profiling, carry out LAMP detection by the detection reagent of embodiment 1 respectively, amplification cycles parameter is: 50 DEG C of preheating 1min; 61 DEG C of reaction 1.5min, run 40 circulations, utilize the accumulation Real-Time Monitoring LAMP reaction process of fluorescent signal, cover the staining agent SYBRGreenI(10000 × of instillation 0.1 μ L at the pipe of reaction tubes), SYBRGreenI must not contact with mixed solution.After amplification terminates, the centrifugal 1min of 8000r/min, makes staining agent fully mix with amplified production, observes coloration result, resulting table agriculture pawpaw, Hainan pawpaw, Thailand pawpaw and Taiwan nonage Fructus Chaenomelis powder fluorescent reaction are positive (safran), belong to transgenic papaya GMYK strain; Other is negative, and do not belong to transgenic papaya GMYK strain, its result is consistent with industry standard SN/T2653-2010 detected result.These pawpaws are different, and some claims to be imported product, and as all happy pawpaw, Thailand pawpaw, happy strange pawpaw do, other claim from Hainan, Guangxi, Taiwan, but its true place of production is difficult to review.
The LAMP specificity of embodiment 4, transgenic papaya GMYK strain and sensitivity test
With the reaction system of embodiment 1 to obtain from above-mentioned purchase and the transgenic papaya GMYK(verified as platform agriculture pawpaw, Hainan pawpaw, Thailand pawpaw or Taiwan nonage Fructus Chaenomelis powder), each supermarket buy non-transgenic papaya, non-transgenic tomato, non-transgenic Radix Dauci Sativae, genetically engineered soybean DNA profiling carry out LAMP amplified reaction, compare their specificity, result transgenic papaya GMYK fluorescent reaction is positive, and other is negative; Illustrate that above-mentioned three pairs of primers have good specificity.10 are carried out to the DNA profiling of transgenic papaya GMYK
0, 10
﹣ 1, 10
﹣ 2, 10
﹣ 3, 10
﹣ 4, 10
﹣ 5, 10
﹣ 6dilute LAMP to detect, result display LAMP detects and is limited to stoste dilution 10 again
-4dilution, i.e. 2.89ng.
<110> Ningbo Institute of Inspection and Quarantine Science Technology
The LAMP detection reagent of <120> transgenic papaya GMYK strain
<160>6
<170>PatentInversion3.5
<210>1
<211>19
<212>DNA
<213> artificial sequence
<220>
<223> is according to the Auele Specific Primer of transgenic papaya GMYK strain gene design
<400>1
CTCATTCAGACCGGTATCC19
<210>2
<211>20
<212>DNA
<213> artificial sequence
<220>
<223> is according to the Auele Specific Primer of transgenic papaya GMYK strain gene design
<400>2
AGCAAGTGGATTGATGTGAT20
<210>3
<211>44
<212>DNA
<213> artificial sequence
<220>
<223> is according to the Auele Specific Primer of transgenic papaya GMYK strain gene design
<400>3
CGGGGGACTCTAGAGGATCCTTTTCAGCTTCATTTTTAGACGCC44
<210>4
<211>48
<212>DNA
<213> artificial sequence
<220>
<223> is according to the Auele Specific Primer of transgenic papaya GMYK strain gene design
<400>4
TCCTTATATAGAGGAAGGGTCTTGCTTTTCCACTGACGTAAGGGATGA48
<210>5
<211>20
<212>DNA
<213> artificial sequence
<220>
<223> is according to the Auele Specific Primer of transgenic papaya GMYK strain gene design
<400>5
GGGTGGTCAGTCCCTTCCAT20
<210>6
<211>21
<212>DNA
<213> artificial sequence
<220>
<223> is according to the Auele Specific Primer of transgenic papaya GMYK strain gene design
<400>6
GAAGGATAGTGGGATTGTGCG21
Claims (1)
1. the LAMP detection reagent of transgenic papaya GMYK strain, comprises 10 × LAMP reaction buffer, trimethyl-glycine, dNTP, BstDNA polysaccharase and three pairs of Auele Specific Primers, it is characterized in that the nucleotide sequence of described three pairs of Auele Specific Primers is respectively:
F3：CTCATTCAGACCGGTATCC
B3：AGCAAGTGGATTGATGTGAT
FIP：CGGGGGACTCTAGAGGATCCTTTTCAGCTTCATTTTTAGACGCC
BIP：TCCTTATATAGAGGAAGGGTCTTGCTTTTCCACTGACGTAAGGGATGA
Loop1：GGGTGGTCAGTCCCTTCCAT
Loop2：GAAGGATAGTGGGATTGTGCG。
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| CN108220397A (en) * | 2018-02-26 | 2018-06-29 | 杭州更蓝生物科技有限公司 | A kind of method for detecting transgenosis pawpaw |
| CN109055592A (en) * | 2018-08-17 | 2018-12-21 | 海南医学院 | A kind of anti-ring spot virus papaya YK16-0-1 of transgenosis and its efficient qualitative, quantitative identification method of spin-off |
| CN114622028B (en) * | 2022-03-07 | 2023-12-22 | 江汉大学 | Primer pair combination, kit and detection method for detecting transgenic papaya |
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Non-Patent Citations (2)
| Title |
|---|
| 抗病毒转基因番木瓜的实时PCR检测;韩建勋等;《检验检疫学刊》;20100131;第20卷(第1期);第15-20页，参见第2.2.2节 引物和探针和图1 * |
| 环介导等温扩增技术检测含有CaMV35S的转基因玉米;陈金松等;《华北农学报》;20110430;第26卷(第4期);第8-14页，参见参见摘要和第1.2.3节LAMP反应及条件的优化 * |
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Inventor after: Chen Xianfeng Inventor after: Zhang Jihong Inventor after: Chen Jiong Inventor after: Zhang Huili Inventor before: Zhang Jihong Inventor before: Yu Shuqiong Inventor before: Chen Xianfeng Inventor before: Zhang Huili Inventor before: Gu Jianfeng |
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Free format text: CORRECT: INVENTOR; FROM: ZHANG JIHONG YU SHUQIONG CHEN XIANFENG ZHANG HUILI GU JIANFENG TO: CHEN XIANFENG ZHANG JIHONG CHEN JIONG ZHANG HUILI |
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Address after: 315000 Block A, Qingyi Road, Ningbo High-tech Zone, Zhejiang Province Patentee after: Ningbo Institute of Inspection and Quarantine Science Technology Address before: 315012 Ma Yuan Road, Haishu District, Ningbo, Zhejiang Province, No. 9 Patentee before: Ningbo Institute of Inspection and Quarantine Science Technology |