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Transformant specific primers and probes of herbicide-tolerant transgenic cotton MON1445, and application in real-time fluorescence PCR detection
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A MON1445 and herbicide-tolerant technology, applied in the field of molecular biology, can solve the problems of insufficiently advanced technology and insufficient sensitivity, and achieve the effects of good versatility, strong specificity, broad application value and market prospect
Inactive Publication Date: 2013-10-23
INST OF PLANT PROTECTION SHANDONG ACAD OF AGRI SCI
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[0005] For the above-mentioned prior art, the object of the present invention is to provide a kind of transformant-specific primers and probes of herbicide-resistant transgenic cotton MON1445 and the application in real-time fluorescent PCR detection, which overcomes the insufficient advanced technology and insufficient sensitivity of the existing methods high disadvantage
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[0034] MON1445-qR: 5'-ACAACATGCATCAATCGACCTG-3'; as shown in SEQ ID No.1, SEQ ID No.2.
[0035]Extract transgenic cotton MON1445 (1%, w / w) and transgenic corn Bt176 (1%, w / w), MON810 (1%, w / w), T25 (1%, w / w), TC1507 (1% ,w / w), transgenic soybean MON87701(1%,w / w), transgenic rice Kefeng 6(1%,w / w), transgenic cotton MON531(1%,w / w) genomic DNA, respectively with MON1445 -qF / MON1445-qR primer combination for real-time fluorescent PCR amplification.
[0036] The extracted transgenic cotton MON1445 (1%, w / w) genomic DNA was sequentially diluted to 1%, 0.1%, 0.01%, and 0% as a template for SYBR Green I real-time fluorescent PCR amplification.
[0037] The amplification reaction volume is 20 μL, 2×FastStart Uni...
[0040] Primers and probes were synthesized by Dalian Takara Company and diluted to 10 μmol L -1 spare. The sequences of the synthesized primers and probes are as follows:
[0043] MON1445-qP: 5'-FAM-CTGAAGGCGGGAAACGACAATCTGATC-TAMRA-3'; shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3.
[0044] Extract transgenic cotton MON1445 (1%, w / w) and transgenic corn Bt176 (1%, w / w), MON810 (1%, w / w), T25 (1%, w / w), TC1507 (1% ,w / w), transgenic soybean MON87701(1%,w / w), transgenic rice Kefeng 6(1%,w / w), transgenic cotton MON531(1%,w / w) genomic DNA, respectively with MON1445 -qF / MON1445-qR / MON1445-qP primer and probe combination for real-time fluorescent PCR amplification.
[0045] The extracted transgenic cotton MON1445 (1%, w / w) genomic DNA was diluted to 1%, 0.1%, 0.01%, 0% in sequence, and used as a t...
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Abstract
The invention relates to transformant specific primers and probes of herbicide-tolerant transgenic cotton MON1445, and an application in real-time fluorescence PCR detection. The sequences of the primers and the probes are shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, the genomeDNA of a to-be detected sample is extracted and added with primers for amplification then for SYBR Green I real-time fluorescence PCR detection; and the genomeDNA of the to-be detected sample is extracted and added with combination of the primers and the probes for amplification then for Taqman probe real-time fluorescence PCR detection. The designed primer and probe sequences of the invention are applied to PCR detection, and the detection is sensitive, accurate, simple and realiable.
Description
technical field [0001] The invention relates to specific primers and probes for herbicide-resistant transgenic cotton MON1445 transformants and their application in real-time fluorescent PCR detection, belonging to the field of molecular biology. Background technique [0002] The PCR detection of transgenic plants and their products is mainly the selective amplification of exogenous inserts in transgenic plants. The specificity and detection range of PCR amplification for different positions and elements of exogenously inserted nucleic acid fragments are very different. According to the position of the amplified target nucleic acid, PCR detection strategies can be divided into four types, namely Screen PCR detection (Screen PCR), gene-specific PCR detection (Gene-Specific PCR), and construct-specific PCR detection (Construct-Specific PCR detection). PCR) and strain-specific PCR detection (Event-Specific PCR). Line-specific PCR detection is realized by detecting the DNA seq...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 李凡张广远孙红炜路兴波雒珺瑜孙廷林
Owner INST OF PLANT PROTECTION SHANDONG ACAD OF AGRI SCI
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