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LAMP detection reagent for transgenic papaya gm YK line

A detection reagent, the technology of papaya, which is applied in the field of reagents for detection of papaya, achieves the effects of easy popularization and application, intuitive results, and low difficulty in judgment

Active Publication Date: 2016-01-20
NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] For the various transgenic papayas that may appear in the market, our commonly used detection methods for genetically modified crops include qualitative PCR, multiplex PCR, real-time fluorescent quantitative PCR, test strips, etc. Inspection and Quarantine Journal 2010 (1) 15-20], these detection methods have different degrees of defects in efficiency, cost and operability

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1, the LAMP detection reagent of transgenic papaya GMYK strain

[0016] In a 25 μL reaction system: 2.5 μL of 10×LAMP reaction buffer, 1.5 μL of BstDNA polymerase at a concentration of 8 U / L, 4.0 μL of dNTP at a concentration of 2.5 mmol / L, and MgCl at a concentration of 25 mmol / L 2 2.5 μL, 4.0 μL of betaine with a concentration of 5.0 mol / L, 0.2 μL each of FIP and BIP with a concentration of 20 μmol / L, 1.6 μL each of F3 and B3 with a concentration of 20 μmol / L, both with a concentration of 20 μmol / L 0.8 μL each for Loop1 and Loop2. LAMP reaction buffer and BstDNA polymerase were purchased from NEWENGLANDBiolabs, dNTP Bioengineering (Dalian) Co., Ltd., and betaine was purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.

Embodiment 2

[0017] Embodiment 2, DNA extraction

[0018] Papaya DNA was extracted according to the operating instructions of the DneasyPlantMiniKit reagent (purchased from QIAGEN, Germany): (1) Weigh 200 mg of the sample, use the bead milling method to homogenize the sample, and put it into a 1.5 mL centrifuge tube; (2) Add 500 μL of buffer AP1, oscillating in a oscillating constant temperature metal bath at 65°C for 15 minutes; (3) Add 130 μL of buffer solution AP2, mix thoroughly by inverting up and down, and let stand in a refrigerator at 4°C for 5 minutes; (4) Centrifuge at 12000 r / min at room temperature for 5 minutes, and take Transfer 500 μL of the supernatant to a 2mL collection tube; (5) Put the collection tube into the QIAcube automatic nucleic acid extractor, select the DNeasyPlantMini mode, automatically extract DNA, and use the U-0080D biospectrophotometer to measure the OD 260 and OD 280 Calculate the DNA concentration; (6) Take out the DNA sample template and store it in a...

Embodiment 3

[0019] Embodiment 3, LAMP detection

[0020] We bought papayas or processed products from Ningbo Supermarket: Hainan papaya, red heart papaya, Dole papaya, Thai papaya, Guangxi green papaya, Risheng papaya, Tainong papaya, Leqi dried papaya, Taiwan green papaya powder, etc. The LAMP detection of the present invention is used for detection. The detection method includes DNA extraction as in Example 2. Take 2 μL of each DNA template, and use the detection reagent of Example 1 to perform LAMP detection respectively. The amplification cycle parameters are: preheating at 50 ° C for 1 min; React at 61°C for 1.5 min and run for 40 cycles. Use the accumulation of fluorescent signals to monitor the LAMP reaction process in real time. Drop 0.1 μL of the dye SYBRGreenI (10000×) on the cap of the reaction tube. SYBRGreenI must not come into contact with the mixture. After the amplification, centrifuge at 8000r / min for 1min to fully mix the dye and the amplification product, and observe th...

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PUM

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Abstract

The invention discloses an LAMP detection reagent for a transgenosis carica papaya GMYK strain. Outer primers F3 and B3, inner primers FIP and BIP and loop primers Loop 1 and Loop 2 are utilized for amplification of target sequences; the LAMP detection reagent has the advantages of high specificity and sensitivity; the key points of the LAMP technology are as follows: as for 6-8 areas of target genes, three pairs of specific primers are designed, a detection reagent for specificity of the antiviral transgenosis carica papaya GMYK strain is created, and the amplification products are processed through the fluorochrome SYBR Green I, so that visualization of an observed result is realized, and whether the transgenosis carica papaya reaches the entry and exit inspection and quarantine standards and whether the transgenosis carica papaya conforms to China's transgenosis carica papaya production requirements are detected. During detection, the operation is simple, only one simple thermostat water bath is needed, the cost is low, and easiness in operation in the field is realized or grass-roots popularization and application are realized.

Description

technical field [0001] The invention relates to a reagent for detecting papaya, in particular to a LAMP detection reagent for transgenic papaya GMYK strain. Background technique [0002] Papaya (Caricapapaya), also known as papaya, is a fruit of the cruciferous papaya family. The United States has been committed to the development of transgenic papaya research against PRSV since 1985. In 1989, the CP gene of PRSV strain HA5-1 was cloned and sequenced, and the gene gun transformation of papaya embryos began. Fitch et al. [FitchMMM, ManshardtRM, GonsalvesD, etal.Stabletransformationofpapayaviamicroprojectilebombardment.PlantCellRep, 1990, 9:189~194] successfully transfected the gene encoding PRSV capsid protein (Coatprotein, CP) into papaya, and obtained the disease resistance of the transgenic CP gene The strain "55-1" was approved for commercial production in 1997. Li Huaping [RuanXL, LiHP, ZhouGH. Evaluation of PRSV resistance of T2 transgenic papaya with replicase gene. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 陈先锋张吉红陈炯张慧丽
Owner NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE