Transgenic maize mon88017 constructs a primer set and probe for accurate detection of specific quantitative PCR and its method

A technology of genetically modified corn and detection methods, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., to achieve the effects of good sensitivity, good specificity, and high amplification efficiency

Inactive Publication Date: 2019-10-29
四川省农业科学院分析测试中心
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method has high amplification efficiency, high accuracy and good sensitivity, and effectively solves the problem of high-sensitivity quantitative PCR precision detection technology for non-transgenic corn MON88017 in the prior art

Method used

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  • Transgenic maize mon88017 constructs a primer set and probe for accurate detection of specific quantitative PCR and its method
  • Transgenic maize mon88017 constructs a primer set and probe for accurate detection of specific quantitative PCR and its method
  • Transgenic maize mon88017 constructs a primer set and probe for accurate detection of specific quantitative PCR and its method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The quantitative detection in this embodiment adopts the 7500 fluorescent quantitative PCR instrument produced by ABI Company.

[0033] The construction of a specific quantitative PCR accurate detection method for transgenic maize MON88017 includes the following steps:

[0034] Step 1: Synthesize upstream primers, downstream primers and fluorescent probes used in conjunction with the primers,

[0035] Upstream primer sequence: Construct MON88017-Forward:

[0036] 5'-GGGCTTAGATGAGAAACTTCACGAT-3';

[0037] Downstream primer sequence: Construct MON88017-Reverse:

[0038] 5'-CCGACTCTCTTCTCAAGCATATGA-3';

[0039] Fluorescent probe sequence: Construct MON88017-Probe:

[0040] 5'-FAM-CGCGCCAAAGCTTACTCGAGGTCA-TAMARA-3'.

[0041] Wherein, the synthetic concentration of upstream primer, downstream primer and fluorescent probe is 10 μmol / l.

[0042] The nucleotide sequences of the above upstream primers, downstream primers and fluorescent probes are designed for a specific s...

Embodiment 2

[0050] Amplification efficiency experiment

[0051] The method data of this embodiment was continuously repeated to make 29 parallel samples, and the 29 samples were detected, and the detection data are shown in Table 1.

[0052] Table 1

[0053]

[0054]

[0055] The invention can accurately detect the specific DNA fragment and its content constructed by MON88017 in transgenic corn, obtain the slope of the standard curve, which is between -3.6 and -3.1, the correlation coefficient is greater than 0.99, and the amplification efficiency is 102.102%, which is 90% to 110%. In the range. The quantitative test result (1.6%) of the sample to be tested is very close to the true value (1.5%), the relative deviation (6.7%) of the test result is less than 25% internationally recognized, and the uncertainty of the test result is less than 10%.

[0056] The above results show that the present invention has good amplification efficiency.

Embodiment 3

[0058] Specific detection

[0059] Materials used to build specificity:

[0060] Transgenic corn: T25, MON810, Bt11, Bt176, GA21, TC1507, 59122, MON89788, NK603, MON863, BvLa;

[0061] Transgenic rice: KF6, KMD, TT51-1, TA2-1;

[0062] Genetically modified tobacco: TMV;

[0063] Transgenic sugar beet: H7-1;

[0064] Genetically modified soybean: GTS40-3-2;

[0065] Transgenic cotton: MON15985, LLC25, MON1531, MON88913, GHB614, MON1445;

[0066] Transgenic rapeseed: GT73, T45, MS8, RF1, RF2, RF3.MS1, MS8

[0067] The DNA of the above-mentioned samples was analyzed using the primer set and probe of the present invention.

[0068] The result is as figure 1 As shown, using the primer set and probe of the present invention to detect transgenic corn non-MON88017 varieties, transgenic rice, transgenic soybean, transgenic rape and non-transgenic corn, rice, rape, soybean (horizontal curve in the figure) and transgenic corn MON88017 strain test materials, Only the material of tra...

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Abstract

The invention provides a primer group and probe for building specific quantitative PCR (polymerase chain reaction) precise detection by transgenic maize MON88017 and a detecting method thereof. The sequence of a forward primer is 5'-GGGCTTAGATGAGAAACTTCACGAT-3'; the sequence of a reverse primer is 5'-CCGACTCTCTTCTCAAGCATATGA-3'; the sequence of the probe is 5'-FAM CGCGCCAAAGCTTACTCGAGGTCA-TAMARA-3'. The primer group and the probe provided by the invention have the advantages that the specificity is good; the specific detection of the transgenic maize MON88017 can be realized; the detection method has the advantages of high amplification efficiency, high accuracy degree and good sensitivity degree. The problem that no high-sensitivity quantitative PCR precise detection technology by the transgenic maize MON88017 exists in the prior art can be effectively solved. The method can be applied to detection of the transgenic maize MON88017 and products of the transgenic maize MON88017.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer set and a probe for constructing specific quantitative PCR accurate detection of transgenic maize MON88017 and a method thereof. Background technique [0002] At present, the detection technology of transgenic maize MON88017 focuses on the common qualitative PCR analysis method, and there is no high-sensitivity quantitative PCR accurate detection technology for the amplification detection of a specific site (gene sequence) specific to the construction of transgenic maize MON88017 and its products. Therefore, providing a quantitative PCR precise detection technology with high amplification efficiency and high accuracy for detecting transgenic maize MON88017 and its products has become an urgent problem to be solved by those skilled in the art. Contents of the invention [0003] The invention provides a primer set and a probe for constructing specific quantitativ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6895C12Q2531/113
Inventor 宋秋池宋君王东雷绍荣郭灵安尹全张富丽常丽娟刘文娟
Owner 四川省农业科学院分析测试中心
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