中文摘要：本申请公开了一种转基因玉米品系MIR162的定量检测方法和试剂。本申请的定量检测方法，包括在PCR反应体系中加矿物油，混匀转移到微滴发生器产生微滴，然后进行PCR反应，结束后，读取每个微滴扩增信号，用QuantaSoft软件计算待测样品的靶标DNA分子数；PCR反应体系中包括MIR162品系和HMG基因特异引物和探针，两条探针分别采用FAM和HEX荧光标记。本申请的定量检测方法，设计配套使用的内源基因HMG和MIR162品系特异性引物探针，采用双通道法，分别定量出两者在玉米基因组中的分子数，计算出转基因玉米品系MIR162绝对含量，对转基因成分的研究和我国口岸转基因检测具有重要意义。
外文摘要：NOVELTY - Quantitative detecting of transgenosis corn strain MIR162 comprises adding oil in polymerase chain reaction system, mixing uniformly, transferring it to droplet generator, generating automatically a droplet, transferring all droplets in the reaction tube to carry out polymerase chain reaction, finishing polymerase chain reaction, reading of each droplet amplification signal, and using QuantaSoft V1.3.2 (RTM: PCR data exporting software) software to calculate sample number of target DNA molecules.
USE - The method is useful for quantitative detecting of transgenosis corn strain MIR162 (claimed).
DETAILED DESCRIPTION - Quantitative detecting of transgenosis corn strain MIR162 comprises adding oil in the polymerase chain reaction system, mixing uniformly, transferring it to the droplet generator, generating automatically a droplet, transferring all the droplets in the reaction tube to carry out polymerase chain reaction, finishing polymerase chain reaction, reading of each droplet amplification signal, and using QuantaSoft V1.3.2 (RTM: PCR data exporting software) software to calculate the sample number of target DNA molecules, where the polymerase chain reaction system is duplication real-time fluorescence polymerase chain reaction amplification system comprises polymerase chain reaction liquid, MIR162 strain specific primers, MIR162 strain specific probe, high-mobility group (HMG) gene specific primer, HMG gene specific probes and the sample DNA, the forward primer of the MIR162 strain comprises sequence of 5'-tgcaaataggctgggaatagtctgtc-3' (SEQ ID NO: 1), the reverse primer of the MIR162 strain comprises sequence of 5'-aaacggcttgtcccgcgtcat-3' (SEQ ID NO:2), the MIR162 strain comprises sequence of 5'-gcgggaaacgacaatctgatcatga-3' (SEQ ID NO: 3), the HMG gene specific primer set of a forward primer comprises sequence of 5'-ttggactagaaatctcgtgctga-3'(SEQ ID NO: 4), the reverse primer of the HMG gene comprises sequence of 5'-gctacata! gggagcct tgtcct-3' (SEQ ID NO: 5), the HMG gene comprises sequence of 5'-caatccacacaaacgcacgcgta-3'(SEQ ID NO: 6). INDEPENDENT CLAIMS are also included for:
(1) transgenic corn strain MIR162 quantitative detection kit comprises a first primer probe and the second primer probe, where the first primer probe for MIR162 strain specificity detection, second primer probe combinations for the HMG gene specific detection, the first primer probe combination forward primer sequence is represented by (SEQ ID NO: 1), and the reverse primer (SEQ ID NO: 2), probe is (SEQ ID NO:3), and the probe 5' end with FAM fluorescent group, the 3 ' end has BHQ-1 quenching group, the second primer probe combination, the forward primer is (SEQ ID NO: 4), reverse primer (SEQ ID NO: 5), probe sequence (SEQ ID NO: 6), and the probe of 5 ' end has a HEX fluorescent group, the 3 ' end has BHQ-1 quenching group;
(2) use of reagent system in preparing transgenic maize detection kit or detection device;
(3) transgenosis corn detecting reagent box comprising the reagent box; and
(4) use of the transgenic corn detection kit in quantitative detection of transgenic maize strain MIR162 by quantitative detection method.
主权项：一种转基因玉米品系MIR162的定量检测方法，其特征在于：包括在PCR反应体系中加入矿物油，混匀后，将其转移到微滴发生器上，自动产生微滴，然后将微滴全部转移到反应管中进行PCR反应，PCR反应结束后，读取每个微滴的扩增信号，并采用QuantaSoft?V1.3.2软件计算待测样品的靶标DNA分子数；所述PCR反应体系为双重实时荧光PCR扩增体系，包括PCR反应液、MIR162品系特异引物组、MIR162品系特异探针、HMG基因特异引物组、HMG基因特异探针和待测样品的DNA；所述MIR162品系特异引物组的正向引物为Seq?ID?No.1所示序列，MIR162品系特异引物组的反向引物为Seq?ID?No.2所示序列，所述MIR162品系特异探针为Seq?ID?No.3所示序列，所述HMG基因特异引物组的正向引物为Seq?ID?No.4所示序列，HMG基因特异引物组的反向引物为Seq?ID?No.5所示序列，所述HMG基因特异探针为Seq?ID?No.6所示序列；Seq?ID?No.1：
申请号：CN201610934341.5
专利号：CN106566878-A
公开/公告号：CN106566878A
申请日：2016-10-25
公开/公告日：2017-04-19
申请/专利权人：深圳出入境检验检疫局动植物检验检疫技术中心
发明/设计人：凌杏园 ; 潘广 ; 叶奕优 ; 龙海 ; 章桂明 ; 程颖慧 ; 向才玉 ; 谢晓露
分类号：C12Q1/68 ; C12N15/11
主分类号：C12Q1/68
法律状态：实质审查
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