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Transgenic tobacco multi-fluorescence PCR (polymerase chain reaction) gene loci and primers and detecting method thereof

A gene locus, multiple fluorescent technology, applied in the field of primers and their detection, multiple fluorescent PCR gene loci in transgenic tobacco, can solve the problems of uneven effects

Pending Publication Date: 2019-07-09
CHINA TOBACCO GUIZHOU IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the types of samples and research methods are very different, and the methods of manual extraction are also widely varied and the differences are significant, so the results are uneven.

Method used

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  • Transgenic tobacco multi-fluorescence PCR (polymerase chain reaction) gene loci and primers and detecting method thereof
  • Transgenic tobacco multi-fluorescence PCR (polymerase chain reaction) gene loci and primers and detecting method thereof
  • Transgenic tobacco multi-fluorescence PCR (polymerase chain reaction) gene loci and primers and detecting method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Example 1 Construction and verification of multiple fluorescent kits for detecting tobacco transgenic components

[0118] (1) Primer probe set: as shown in Table 2, it can be synthesized by a company with primer and probe synthesis capabilities. In this example, Shanghai Sangon Bioengineering Technology Service Co., Ltd. was selected to synthesize the primer and probe dry powder. 100 μmol / L was used as the stock solution, and 10 μmol / L was prepared as the use solution according to Table 1.

[0119] Table 2 Preparation of 100μmol / L stock solution into 10μmol / L use solution

[0120]

[0121]

[0122] *: packaged in 1.5mL transparent centrifuge tubes; #: packaged in 1.5mL black centrifuge tubes respectively.

[0123] (2) Fluorescent PCR reagents: common commercially available fluorescent PCR reagents, selected in this embodiment Path-ID TM qPCR Master Mix.

[0124] (3) Positive control: Take all primer stock solutions and dilute to 10 μmol / L respectively. Extr...

Embodiment 2

[0141] Example 2 Detection of three transgenic tobacco strain samples of Btc / CpTI / EPSPS, CMV-cp / PVY-cp / TMV-cp, CMV-sat / PVY-Nib / TMV-54kD

[0142] The seeds and fresh leaves of three transgenic tobacco lines, Btc / CpTI / EPSPS, CMV-cp / PVY-cp / TMV-cp, CMV-sat / PVY-Nib / TMV-54kD, were donated from Guizhou Academy of Agricultural Sciences, and they were edited in turn as #1, #2, #3 samples. After the sample was ball milled, DNA was extracted on a small magnetic stand (QiagenMagAttract Magnetic Rack) using an outsourced DNA extraction kit (QiagenMagAttract Hmw DNA kit); the DNA purity was OD260 / OD280=1.79, and the concentrations were 182.7ng / μL, 150.3ng / μL, 194.6ng / μL, stored at -20°C. Afterwards, the DNA was diluted to 50 ng / μL for detection. The primer set, positive control, negative control, and blank control were all in accordance with the kit components in Example 1. The purchased fluorescent PCR reagent was Multiplex PCR Kit, on-machine detection (ABI fluorescence PCR instrument...

Embodiment 3

[0156] Example 3 Detection of Non-transgenic Tobacco Strain Samples

[0157] Received a non-GMO tobacco sample (roasted tobacco leaf powder) from Guizhou Quality Inspection Institute. (1) The sample was pulverized with a ball mill, and the mass after treatment was 10.0g; (2) Add 10mL of n-hexane and 20mL of CTAB extract, homogenize in an air shaker at 120rpm for 6h, and mix by inverting occasionally; (3) 10k rpm, Centrifuge for 10 minutes to make the aqueous phase and organic phase clearly separated, remove the organic phase and pipette the aqueous phase; (4) centrifuge the obtained aqueous phase at 10k rpm for 10 minutes, and pipette the supernatant aqueous phase; (5) use a large-volume DNA extraction kit (QiagenDNeasy Plant Maxi Kit) for DNA extraction; (6) Concentrate the DNA sample to 10 μL through a column membrane concentration tube (Millipore Microcon DNAfast flow (PCR grade)), the DNA purity is OD260 / OD280=1.71, and the DNA concentration is 8.60ng / μL, store at -20°C....

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Abstract

The invention discloses transgenic tobacco multi-fluorescence PCR gene loci and primers and a detecting method thereof. The transgenic tobacco multi-fluorescence PCR gene loci and primers comprise loci, primers and probes of genes of Nr, Btc, CpTI, EPSPS, CMV-cp, PVY-cp, TMV-cp, CMV-sat, PVY-nib and TMV-54D. According to the transgenic tobacco multi-fluorescence PCR gene loci and primers, endogenous and specific exogenous genes of transgenic tobacco lines are simultaneously applied to design of specific primer-probe groups, and the primers and the probes can avoid mutual interference and can only perform amplification and fluorescence signals on specific target sequences but have no amplification or fluorescence signals on non-targeted sequences, so that high specificity and detecting sensitivity can be achieved.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a transgenic tobacco multiple fluorescent PCR gene locus, primers and a detection method thereof. Background technique [0002] In 1983, the world’s first genetically modified crop, that is, genetically modified 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; caused numerous controversies. [0003] The relevant regulations on genetically modified tobacco issued by our country include Guoyanfa [1998] No. 168 "Tobacco Genetic Engineering Research and Its Application Management Measures", Guoyanke [2003] No. 387 "Notice of the State Tobacco Monopoly Administration on Strengthening the Monitoring of Genetically Modified Tobacco" 》and several documents on strengthening genetically modified testing of exported tobacco were succe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11C12Q1/686
CPCC12Q1/6895C12Q1/686C12Q2537/143C12Q2563/107Y02A50/30
Inventor 董睿刘剑王维维陈永芳张丽胡硕万强
Owner CHINA TOBACCO GUIZHOU IND
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