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Specific real-time fluorescent PCR (Polymerase Chain Reaction) detection primer, probe, method and kit for transgenic potato AM04-1020

A technology for detecting primers and real-time fluorescence, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc.

Pending Publication Date: 2022-04-15
TECH CENT OF GUANGZHOU CUSTOMS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no specific real-time fluorescent PCR detection method for transgenic potato AM04-1020 in other countries and regions such as the European Union

Method used

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  • Specific real-time fluorescent PCR (Polymerase Chain Reaction) detection primer, probe, method and kit for transgenic potato AM04-1020
  • Specific real-time fluorescent PCR (Polymerase Chain Reaction) detection primer, probe, method and kit for transgenic potato AM04-1020
  • Specific real-time fluorescent PCR (Polymerase Chain Reaction) detection primer, probe, method and kit for transgenic potato AM04-1020

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Establishment of real-time fluorescent PCR detection method

[0044] According to the specific sequence of transgenic potato AM04-1020 (shown as SEQ ID NO.7), primers and probes were designed using PrimerPremier 5.0 software. Potato ST-LS1 is used for the detection of DNA in potato-derived samples and the relative quantification of transgenic components. The specific primers and probes are listed in Table 1.

[0045] Table 1 Primers and probes for real-time fluorescent PCR

[0046]

[0047] The amplification reaction system is 25μL: including Premix Ex Taq TM 12.5 μL, ROX Reference Dye II 0.2 μL, 10 μmol / L upstream primer 0.5 μL, 10 μmol / L downstream primer 0.5 μL, 10 μmol / L detection probe 0.5 μL, DNA template 2 μL and ddH 2 O 8.8 μL; the reaction program was 95°C for 30s; 95°C for 5s, 60°C for 34s, 40 cycles, and the fluorescence signal was collected at 60°C.

Embodiment 2

[0048] Embodiment 2: real-time fluorescent PCR method specificity test

[0049] Transgenic potato EH92-527-1, non-transgenic potato, transgenic corn MIR162, transgenic alfalfa J163×J101, transgenic soybean DAS44406-4, transgenic soybean DAS81419-2, transgenic soybean DAS68416, transgenic soybean GTS40-3-2, transgenic rape MON88302 , transgenic rapeseed DP073496-4, transgenic maize MON98034, transgenic cotton MON15985-7, non-transgenic rice, wheat, non-transgenic sugar beet genomic DNA as templates, and the positive sample was AM04-1020 sample DNA. The specificity of the established AM04-1020 transgenic potato real-time fluorescent PCR detection method was tested. The real-time fluorescent PCR detection reaction system and reaction procedure are the same as in Example 1.

[0050] When using AM04-1020 transgenic potato-specific primer AM04-1020-5F2 / 5R2 and probe AM04-1020-3P (see Table 1 for specific primer sequences) to carry out real-time fluorescent PCR on the DNA of all ext...

Embodiment 3

[0051] Embodiment 3: Sensitivity test, repeatability test and standard curve establishment

[0052] Dilute the extracted AM04-1020 transgenic potato DNA solution plus TE buffer to 10%, 2.5%, 0.5%, 0.25%, 0.05%, 0.025% and 0.01% (corresponding to copy number 2777.70copies / μL, 694.43copies / μL , 138.89 copies / μL, 69.44copies / μL, 13.89copies / μL, 6.94copies / μL, 2.78copies / μL, based on the weight of potato genome 1.8pg) as DNA template, real-time fluorescent PCR detection of AM04-1020 transgenic potato was carried out. For the linear range test and repeatability test, each sample was repeated three times, water was used as a blank control, and the real-time fluorescent PCR detection reaction system and reaction procedure were the same as in Example 1. The test results show (Table 2 and figure 2 ), within 10%, 2.5%, 0.5%, 0.25%, 0.05%, 0.025%, 6 concentration gradients of 1-6 and 3 parallels can have typical amplification curves ( figure 2 ); In addition, the SD of each 3 paralle...

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Abstract

The invention discloses an AM04-1020 transgenic potato specificity real-time fluorescence PCR (Polymerase Chain Reaction) detection primer, a probe, a method and a kit. The primer and the probe are designed according to the specific sequence of the AM04-1020 transgenic potato, the specific real-time fluorescent PCR detection method for the AM04-1020 transgenic potato is established, the method has the advantages of being good in specificity, high in sensitivity, good in repeatability and the like, the amplification efficiency is 94.39%, and the lowest quantitative detection limit is 0.025%. The problem that no AM04-1020 transgenic potato specificity accurate quantitative detection technology exists in the prior art is effectively solved, and the method can be applied to detection of AM04-1020 transgenic potatoes and products of the AM04-1020 transgenic potatoes in entry and exit port inspection and quarantine and domestic agricultural product food supervision.

Description

technical field [0001] The invention belongs to the field of transgenic product detection, and in particular relates to a primer probe, method and kit for real-time fluorescent PCR detection of transgenic potato AM04-1020. Background technique [0002] At present, there are about 47 kinds of transgenic potatoes on the market abroad (ISSSA data). Among the strains currently on the market abroad, only the EH92-527-1 strain established by the European Union. Strain-specific detection methods: Savini C., Foti N., Mazzara M., Charles Delobel C., Van Den Eede G.; "Event-specific Method for the Quantification of Event EH92-527-1Potato Using Real-time PCR-Validation Report and Protocol-SAMpling and DNA Extraction of Potato" Online Publication(2006); In addition, Gao Hongwei An unmarketed potato line av43-6-g7 detection method established by et al.: (Gao H, Yu X, Deng T, et al.Event-specific detection of transgenic potato AV43-6-G7 using real-time and digitalPCR methods[ J]. Bmc Bio...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/6851C12N15/11
CPCC12Q1/6895C12Q1/686C12Q1/6851C12Q2561/113C12Q2563/107C12Q2561/101C12Q2531/113C12Q2545/113
Inventor 刘二龙卢丽关丽军王毅谦郑冠津魏霜李志勇
Owner TECH CENT OF GUANGZHOU CUSTOMS
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