Multiple DPO-PCR primer combination for detecting transgenic maize MON810 and MIR604 and method

A DPO-PCR, MON810-F technology, applied in the field of multiplex DPO-PCR primer combinations, can solve the problems of complex system, low stability, limited use, etc., and achieve high throughput, good specificity, and improved detection efficiency. Effect

Active Publication Date: 2015-12-02
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Due to the addition of multiple pairs of primers in the PCR reaction system, the system is complex and the stability is not high, which limits its use

Method used

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  • Multiple DPO-PCR primer combination for detecting transgenic maize MON810 and MIR604 and method
  • Multiple DPO-PCR primer combination for detecting transgenic maize MON810 and MIR604 and method
  • Multiple DPO-PCR primer combination for detecting transgenic maize MON810 and MIR604 and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Multiplex DPO-PCR method for detecting transgenic maize MON810 and MIR604

[0030] 1. Design of Multiplex DPO-PCR Primer Combinations

[0031] According to the flanking sequences of transgenic maize MON810 and MON810, the following multiple DPO-PCR primer combinations for detecting transgenic maize MON810 and MIR604 were designed (I in the primer sequence is hypoxanthine):

[0032] MON810-F: 5'-CTAACGTTTAACATCCTTTGCCATTIIIIIGCTATCTGTC-3' (SeqIDNo.1)

[0033] MON810-R: 5′-TAGTGGGATTGTGCGTCATCCCTIIIIIIICAGTGGAGA-3′ (SeqIDNo.2)

[0034] MIR604-F: 5′-GCACGCAATTCAACAGAAGGCGIIIIICGACAATC-3′ (SeqIDNo.3)

[0035] MIR604-R: 5'-TGCGGTTCTGTCAGTTCCAAACGTIIIIIIGGCTTGTC-3' (SeqIDNo.4)

[0036] Among them, MON810-F and MON810-R are primers for detecting transgenic maize MON810, and the product size is 266bp; MIR604-F and MIR604-R are primers for detecting transgenic maize MIR604, and the product size is 127bp.

[0037] 2. Multiplex DPO-PCR method for detection of transge...

Embodiment 2

[0052] The specific detection of embodiment 2 multiple DPO-PCR primers

[0053] 1. Extraction of DNA

[0054] Refer to the kit instructions to operate (Plant Genomic DNA Extraction Kit, Cat. No. DP305-02, Tiangen Biotechnology Co., Ltd.) to extract transgenic corn MON810, genetically modified corn MIR604, genetically modified corn TC1507, genetically modified corn MON863, genetically modified corn BT176, and genetically modified corn Genomic DNA of MON89034, transgenic soybean GTS40-3-2, transgenic rice TT51-1, and transgenic rapeseed GT73.

[0055] 2. Multiplex DPO-PCR amplification

[0056] Using the method for detecting transgenic maize MON810 and MIR604 in step 2 of Example 1, the genomic DNA of the nine transgenic lines extracted in step 1 was used as a template for multiplex DPO-PCR amplification to obtain multiplex DPO-PCR amplification products.

[0057] 3. Electrophoretic detection of multiplex DPO-PCR amplification products

[0058] After the multiplex DPO-PCR rea...

Embodiment 3

[0063] The sensitivity detection of embodiment 3 multiplex DPO-PCR primer combination

[0064] 1. Extraction of DNA

[0065] Genomic DNA of transgenic corn MON810 and MIR604 were extracted according to the instructions of the kit (Plant Genomic DNA Extraction Kit, Cat. No. DP305-02, Tiangen Biotechnology Co., Ltd.). And the obtained genomic DNA was diluted 10 times, and the genomic DNA of the transgenic maize 810 with the concentration of 50ng / μL, 5ng / μL, 0.5ng / μL, 0.05ng / μL and 0.005ng / μL and the concentration of 50ng / μL were prepared respectively. μL, 5ng / μL, 0.5ng / μL, 0.05ng / μL and 0.005ng / μL of genomic DNA of transgenic maize MIR604.

[0066] 2. Multiplex DPO-PCR amplification

[0067] Using the method for detecting transgenic maize MON810 and MIR604 in step 2 of Example 1, the following 5 groups of genomic DNA were used as templates to perform multiple DPO-PCR amplification respectively to obtain multiple DPO-PCR amplification products:

[0068] Group 1: 50 ng / μL of tr...

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Abstract

The invention provides a multiple DPO-PCR primer combination for detecting transgenic maize MON810 and MIR604. Sequences of the primer combination are respectively shown in Seq ID NO. 1-4. The invention also provides a multiple DPO-PCR method for detecting transgenic maize MON810 and MIR604 on the basis of the primer combination. Qualitative detection of transgenic crop samples can be implemented. An experiment shows that the method is good in specificity and high in flux, and is speedy; the detecting efficiency is improved; and the method is an effective method for detecting the transgenic maize MON810 and MIR604.

Description

technical field [0001] The invention relates to molecular biology detection technology, in particular to a combination of multiple DPO-PCR primers and a method for detecting transgenic maize MON810 and MIR604. Background technique [0002] Multiplex PCR refers to adding multiple pairs of primers in one reaction tube to simultaneously amplify multiple target genes, which can achieve high-throughput detection of samples, and has been widely used in the detection of genetically modified species. Due to the addition of many pairs of primers in the PCR reaction system, the system is complicated and the stability is not high, which limits its use. The main principle of DPO (Dualpriming oligonucleotide) primer technology is that its primers contain two independent specific primer regions. The two bases are used to guide the specific extension of the PCR reaction. These two independent specific regions are connected by oligomeric hypoxanthine (Inosine, I). Since the annealing tempe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 付伟魏霜杜智欣王晨光乾义柯张娜刘中勇朱水芳
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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