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Triple fluorescent PCR primer set, probe set, kit and method for detecting transgenic components of pepper

A technology of transgenic components and primer sets, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the unreported multiple fluorescent PCR method for detecting transgenic components of peppers, core primers of pepper transgenic components and Problems such as differences in probe sequences and fluorescent labels, to achieve the effect of saving reagent costs, saving detection time, and good specificity

Active Publication Date: 2020-11-17
贵州省产品质量检验检测院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The prior art has not reported the multiplex fluorescent PCR method for the detection of genetically modified ingredients in peppers, and the reported detection of genetically modified soybeans and tomato, etc., and the detection of genetically modified peppers have significant differences in the core primer and probe sequences and fluorescent labels.

Method used

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  • Triple fluorescent PCR primer set, probe set, kit and method for detecting transgenic components of pepper
  • Triple fluorescent PCR primer set, probe set, kit and method for detecting transgenic components of pepper
  • Triple fluorescent PCR primer set, probe set, kit and method for detecting transgenic components of pepper

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Construction and verification of a triple fluorescent kit for detecting genetically modified components of pepper

[0059] ①Primer set and probe set: As shown in Table 1 and Table 2, they can be synthesized by a company with the ability to synthesize primers and probes. In this example, Shanghai Sangon Bioengineering Technology Service Co., Ltd. was selected to synthesize the primers 1 to 12. The sequences are shown in Seq.ID No.1 to Seq.ID No.12 respectively, and the sequences of probes 13 to 18 are shown in Seq.ID No.13 to Seq.ID No.18 respectively. Dilute the dry powder of primers and probes to 100 μmol / L as the stock solution, and prepare 10 μmol / L as the use solution according to Table 3.

[0060] Table 3 Preparation of 100μmol / L stock solution into 10μmol / L use solution

[0061]

[0062] ②Fluorescent PCR reagents: common commercially available fluorescent PCR reagents, selected in this example Path-ID TM qPCR Master Mix.

[0063] ③ Positive cont...

Embodiment 2

[0079] The detection of embodiment two CMV and TMV double antibody strain line capsicum sample

[0080] We received CMV and TMV double-antibody line peppers from Guizhou Academy of Agricultural Sciences. After ball milling the samples, an outsourced DNA extraction kit (ABI Magmax TMDNA Isolation Kit) in ABI Magmax TM Sample DNA was extracted on Express-96 magnetic bead extraction apparatus; DNA purity was OD 260 / OD 280 =1.80, the concentration is 107.86ng / μL, and stored at -20°C. Afterwards, the DNA was diluted to 100ng / μL for detection, and the primer set, probe set, fluorescent reagent, positive control, negative control, blank control, etc. were all tested on the machine according to the kit components in Example 1 (ABI fluorescent PCR instrument, The model is Step one plus).

[0081] The test results are shown in Table 8: ①Quality control standard: CaATL1, CaMV35S, NPT II, ​​NOS, CMV, and TMV in the positive control all have logarithmic growth in fluorescence, and t...

Embodiment 3

[0084] The detection of embodiment three CMV resistant strain capsicum samples

[0085] Purchasing CMV-resistant strains of peppers, ball milling the samples, and using an outsourced DNA extraction kit (QiagenMagAttract Hmw DNA kit) to extract sample DNA on a small magnetic stand (Qiagen MagAttract Magnetic Rack), the DNA purity is OD 260 / OD 280 =1.79, the concentration is 126.97ng / μL, and stored at -20°C. Primer set, probe set, positive control, negative control, blank control, etc. are all according to the kit components in Example 1, and the purchased fluorescent PCR reagent is Multiplex PCR Kit, on-machine detection (ABI fluorescent PCR instrument, model is Step one plus).

[0086] The test results are shown in Table 9: ①Quality control standard: CaATL1, CaMV35S, NPT II, ​​NOS, CMV, and TMV in the positive control all have logarithmic growth in fluorescence, and the Ct value is ≤30.0, and the negative control and blank control have no fluorescence signal and fluorescen...

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Abstract

The invention discloses a triple fluorescence PCR primer set for detecting pepper transgenic ingredients. The triple fluorescence PCR primer set comprises a triple primer set A for CaATL1, CaMV35S and NPT II genes and a triple primer set B for NOS, CMV and TMV genes and is specifically shown as Seq. ID No.1 to Seq. ID No.12. The invention belongs to the technical field of genetic engineering detection; all the primers and probes cannot mutually interfere; a specific target sequence is amplified and excites a fluorescence signal, the non-target sequence is not amplified and generates no fluorescence signal and accurate detection for the pepper transgenic ingredients can be realized; the triple fluorescence PCR primer set has the advantages of high specificity, low standard error, short detection time, capability of saving reagent cost and the like, and is suitable for the detection for the transgenic ingredients of the pepper product.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering detection, and in particular relates to a triple fluorescent PCR primer set, a probe set, a kit and a method for detecting transgenic components of pepper. Background technique [0002] People depend on food, and food safety is one of the basic pillars of social harmony. In 1983, the world's first genetically modified crop (tobacco) was successfully developed in the United States; in 1994, the extended-ripening and fresh-keeping transgenic tomatoes of the American Monsanto Company were approved for marketing in the United States. At present, the variety and output of genetically modified crops are growing rapidly: the EU has basically banned the cultivation of genetically modified crops, but more imports, processing, and sales, such as corn, rapeseed, soybeans, and sugar beets are all circulated and eaten in the market; the United States has planted and produced a large number of And ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/16C12Q2600/166C12Q2537/143C12Q2563/107C12Q2545/113
Inventor 孙端方罗绍楠戴奕杰董睿李春宇田志强刘廷菊陈梅
Owner 贵州省产品质量检验检测院
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