PCR (polymerase chain reaction) primer composition to detect transgenic soybean line MON87705 and application thereof

A technology of MON87705 and transgenic soybeans, which is applied in DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as large limitations and difficulty in achieving standard concentration gradients, and achieve good specificity

Inactive Publication Date: 2019-07-05
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the quantitative detection of genetically modified ingredients generally uses real-time fluorescent PCR technology, which calculates the content of genetically modified ingredients by drawing standard substance concentration curves and detecting thresholds, but there are great limitations in practical applications, the most important being the preparation of standard curves. First of all, it is difficult to achieve an accurate standard concentration gradient. Secondly, errors will inevitably occur during the operation steps and curve drawing process. Digital PCR can avoid this well and can achieve accurate concentration without being affected by the amplification efficiency. Quantitative

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  • PCR (polymerase chain reaction) primer composition to detect transgenic soybean line MON87705 and application thereof
  • PCR (polymerase chain reaction) primer composition to detect transgenic soybean line MON87705 and application thereof
  • PCR (polymerase chain reaction) primer composition to detect transgenic soybean line MON87705 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1, Digital PCR Quantitative Detection Method for Exogenous Genes of Transgenic Soybean MON87705 Line

[0043] 1. Design of primers and probes

[0044] According to the specific sequence of the transgenic soybean MON87705 strain (that is, the sequence at both ends of the insertion site of the foreign gene at the 5' end of the soybean genome), several different PCR-specific primer pairs and specific probes matching the amplification products of each primer pair were designed, At the same time, a PCR-specific primer pair of the soybean internal reference gene Lectin and a specific probe matching the amplification product of the primer pair were designed. The two probes for different target genes used different reporter fluorescent groups FAM and VIC, and the same The quencher group is labeled with BHQ1; the sequence information of each primer and probe is shown in Table 1.

[0045] Table 1. Primer and probe sequences

[0046]

[0047]

[0048] 2. Quantitati...

Embodiment 2

[0056] Embodiment 2, specific detection

[0057] Different from conventional PCR and qPCR, the specificity can be observed through electrophoretic bands and melting curves respectively. Digital PCR observes the end-point fluorescence in each system. Regardless of whether the PCR product is a specific product, as long as the fluorescence signal is strong enough, it will also be Interpreted as a positive result. Therefore, the primer specificity requirements of dPCR are extremely high. This embodiment uses the optimal combination in Example 1: primer pair B1-F / R and its probe B1 and primer pair A-F / R and its probe A, according to Example 1 In the ddPCR detection method, the genomic DNA of five different transgenic soybean lines MON87705, MON87701, MON87769, DP356043, and DAS68416 standards were detected.

[0058] Result: if figure 2 As shown, the exogenous gene in the standard product of the transgenic soybean line MON87705 has amplified signal, but there is no signal of exog...

Embodiment 3

[0059] Embodiment 3, sensitivity detection

[0060] For the detection of genetically modified ingredients (ie, exogenous genes), the detection of genetically modified ingredients with a low percentage content is the focus and difficulty of the detection of genetically modified ingredients. This example studies the lower limit of double digital PCR detection.

[0061] The genomic DNA of the 50ng / μL transgenic soybean line MON87705 standard was diluted with water, and the relative mass percentages were 100%, 50%, 25%, 5%, 0.5% and 0.05% of the transgenic soybean samples, using the most Excellent combination: primer pair B1-F / R and its probe B1 and primer pair A-F / R and its probe A, according to the ddPCR reaction system and conditions of Example 1, the sensitivity detection of double digital PCR was carried out.

[0062] Results: The quantitative results of the transgenic components of MON87705 are shown in Table 3, and the results of the linear fitting standard curve are shown...

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Abstract

The invention discloses a PCR (polymerase chain reaction) primer composition to detect transgenic soybean line MON87705 and application thereof. The primer composition comprises a primer pair to specifically detect transgenic soybean line MON87705 exogenous gene, including an upstream primer shown as SEQ ID No. 1, and a downstream primer shown as SEQ ID No. 2. The primer composition also comprisesa probe to specifically detect transgenic soybean line MON87705 exogenous gene, the probe has a sequence of SEQ ID No. 3 or its complementary sequence. The primer composition has good specificity andhas sensitivity (lower detection limit) which is the concentration of 0.025 ng / mu L of a DNA template in a PCR amplification reaction system, when applied to detect a sample under test by means of micro-drop digital PCR. The primer composition herein has important application value in the qualitative and quantitative detection of transgenic soybean line MON87705 exogenous gene components.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a PCR primer combination and application for detecting transgenic soybean strain MON87705. Background technique [0002] Genetically modified crops have been commercialized for more than 20 years. In 2016, the planted area reached 185.1 million hectares, and more than 26 countries have planted genetically modified crops. As one of the earliest transgenic plant species to be commercially applied, transgenic soybean has a planting area of ​​91.4 million hectares, accounting for 50% of the total transgenic crops. Genetically modified soybeans have large planting areas in the United States, Brazil and Argentina, and occupy an important position in the international agricultural product market. With the large-scale planting and consumption of genetically modified soybeans around the world, my country is facing various situations and problems: genetically modified crops may be mixed with ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/166
Inventor 付伟刘晓朱鹏宇朱水芳
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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