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Kit for detecting double-stranded RNA of RNAi-based transgenic plants and application of kit

A technology of transgenic plants and kits, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as barren fields, hazards of higher-end species, damage of non-target organisms such as earthworms, etc.

Pending Publication Date: 2020-07-03
SHANGHAI INST OF MEASUREMENT & TESTING TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional methods such as chemical insecticides, although the effect is ideal, can also cause harm to non-target organisms such as earthworms, and because they are difficult to metabolize, long-term use will lead to barren fields and even accumulate in the food chain. Species produce harm

Method used

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  • Kit for detecting double-stranded RNA of RNAi-based transgenic plants and application of kit
  • Kit for detecting double-stranded RNA of RNAi-based transgenic plants and application of kit
  • Kit for detecting double-stranded RNA of RNAi-based transgenic plants and application of kit

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Embodiment 1

[0051] The design and synthesis of embodiment 1 primer probe

[0052] For the double-stranded RNA that triggers specific gene silencing in RNAi transgenic corn DBN11061, design dPCR forward and reverse primers and probes, as shown in Table 1, the nucleotide sequence of the DBN11061 double-stranded RNA forward primer is shown in SEQ ID NO:1 , the nucleic acid sequence of the reverse primer is shown in SEQ ID NO:2, the nucleic acid sequence of the probe is shown in SEQ ID NO:3, synthesized by Thermo Fisher Scientific (China) Co., Ltd.

[0053] Table 1 Primer and Probe Sequences

[0054]

Embodiment 2

[0055] Example 2 Method optimization based on RNAi transgenic corn total RNA extraction

[0056] During the extraction process, the leaves are first ground into powder using liquid nitrogen, and then different kits PureLink TM RNA Mini Kit (ET-Kit1, Thermo Fisher Scientific (China) Co., Ltd.), Column Plant Total RNA Extraction and Purification Kit (ET-Kit2, Sangon Bioengineering (Shanghai) Co., Ltd.) and MiniBEST Plant RNAExtraction Kit (ET-Kit3, Treasure Bioengineering (Dalian) Co., Ltd.) was used to extract total RNA from the leaves of RNAi-based transgenic maize, and the obtained RNA was stored at -80°C. cDNA, and follow QuantStudio TM The default program of the 3D digital PCR instrument was used to perform digital PCR and compare the results. The experiments were repeated 3 times.

[0057] The principles of different plant total RNA extraction kits are basically similar. The extraction process mainly includes: (1) adding liquid nitrogen to fully grind to completely bre...

Embodiment 3

[0060] Example 3 Method optimization for reverse transcription of RNA into cDNA

[0061] Transgenic maize leaf RNA obtained using ET Kit 3 was obtained using different kits, ProtoScript cDNA First Strand Synthesis Kit (RT Kit A, NEB (Beijing) Co., Ltd.), PrimeScript TM II1st Strand cDNA Synthesis Kit (RT Kit B, Treasure Bioengineering (Dalian) Co., Ltd.) and Maxima H Minus First Strand cDNA Synthesis Kit (RT Kit C, Thermo Fisher Scientific (China) Co., Ltd.) for reverse transcription, obtained cDNA according to QuantStudio TM The 3D default program was used to perform digital PCR and compare the results, and the experiments were repeated 3 times.

[0062] Reverse transcription refers to the process of using RNA as a template to synthesize the first strand of cDNA under the action of reverse transcriptase, specifically: (1) react at 65°C for different times, and cool on ice to denature the RNA and open its second strand. (2) add random primers, dNTPs, buffer (buffer) and re...

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Abstract

The invention provides a kit for detecting double-stranded RNA of RNAi-based transgenic plants and application of the kit. The kit comprises a primer and probe composition, primers comprise nucleic acid sequences as shown in SEQ ID NO: 1-2, a probe comprises a nucleic acid sequence as shown in SEQ ID NO: 3. For double-stranded RNA of a transgenic maize line based on an RNAi technology, the invention designs the primers and the probe, and establishes a set of reverse transcription chip-type digital PCR quantitative detection method. The absolute quantification limit of the method is 2.5 copies / [mu]L, and the RSD is 12.4%; and when a low-concentration actual sample is detected to reach 21.7 copies / [mu]L, the relative deviation is 2.7%, and the RSD is 14.6% and meets the requirement that theinternational transgenic quantitative result RSD is smaller than or equal to 25%. The method is used for quantitative detection of transgenic plants based on the RNAi technology, and provides an accurate and reliable technical means for safety evaluation of related transgenic plants in China.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and relates to a kit for detecting double-stranded RNA based on RNAi transgenic plants and its application, in particular to a primer probe composition, kit and detection method for detecting double-stranded RNA of RNAi transgenic corn strains and its application. Background technique [0002] In recent years, with the development of biotechnology and related professions, the research and application of genetically modified crops have become more and more extensive. By the end of 2018, the global planting area of ​​genetically modified crops had reached 191.7 million ha 2 ; At the same time, various countries and regions are increasingly strict on the safety management of genetically modified crops, and have begun to implement labeling systems and systematic management. [0003] RNA interference (RNAi, RNA interference), as an emerging popular transgenic biotechnology, often refer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6851
CPCC12Q1/6895C12Q1/6851C12Q2600/158C12Q2600/13C12Q2521/107C12Q2531/113
Inventor 杨镇州许丽刘刚闻艳丽杨雪王乐乐
Owner SHANGHAI INST OF MEASUREMENT & TESTING TECH
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