中文摘要：本发明公开一种针对转基因大豆DAS44406的内外源基因的双重巢式荧光定量PCR检测的引物和探针组合。本发明将双重PCR、巢式PCR和荧光定量PCR结合起来，对引物和探针的设计进行了严格的控制，并对检测流程进行了改进，建立了一套双重巢式荧光定量PCR检测体系，该体系能够定性和定量检测转基因大豆DAS44406品系特异性序列和大豆内源基因Lectin，该方法灵敏度相比普通荧光定量PCR提高了1个数量级。本发明的方法具有模板需要量少，灵敏度高，通量高，特异性强等优点；能应用于转基因的快速定量检测，为快速、准确、高通量、定量检测转基因提供新方法。
外文摘要：NOVELTY - A primer-probe combination comprises primer (A1), primer (A2)-probe (B1) combination, primer (A3) and primer (A4)-probe (B2) combination, is new.
USE - The primer-probe combination is useful for detecting or auxiliary detection of transgenic soybean DAS44406 (claimed) and soybean endogenous gene Lectin.
ADVANTAGE - The primer-probe combination has high sensitivity, high throughput and the detection method is rapid, accurate and suitable for quantitative detection of transgenic plants.
DETAILED DESCRIPTION - A primer-probe combination comprises primer (A1), primer (A2)-probe (B1) combination, primer (A3) and primer (A4)-probe (B2) combination, is new, where the primer (A1) comprises (SEQ ID NO: 1) and (SEQ ID NO: 2), the primer (A2) comprises (SEQ ID NO: 3) and (SEQ ID NO: 4), the probe (B1) is (SEQ ID NO: 5), the primer (A3) comprises (SEQ ID NO: 6) and (SEQ ID NO: 7), the primer (A4) comprises (SEQ ID NO: 8) and (SEQ ID NO: 9) and the probe (B2) is (SEQ ID NO: 10). gcgcaccctacagattccagt (SEQ ID NO: 1) tgtacggctaagagcgaat (SEQ ID NO: 2), tcagccaattttagacaa (SEQ ID NO: 3), aatttaattactaaaacgc (SEQ ID NO: 4), aaaatattgacgcttagaca (SEQ ID NO: 5), cgctattgtgacctcctcg (SEQ ID NO: 6), gtggaggcatcataggtaat (SEQ ID NO: 7), ccagcttcgccgcttccttc (SEQ ID NO: 8), gaaggcaagcccatctgcaagcc (SEQ ID NO: 9), cttcaccttctatgcccctgaca (SEQ ID NO: 10). INDEPENDENT CLAIMS are included for the following:
(1) kit comprising the primer-probe composition; and
(2) method of detecting or auxiliary detection of transgenic soybean DAS44406 using double nested fluorescent PCR involves using the primer-probe combination or the kit.
TF   TECHNOLOGY FOCUS - BIOTECHNOLOGY - Preferred Composition: The kit further comprises mix (M2) comprising deoxynucleotide (dNTP), reaction buffer and magnesium chloride and mix (M1) comprising DNA polymerase, optionally mix (M2) and mix (M1) are reagent kit RR060A obtained from Bao biological engineering Dalian company limited, where the kit optionally comprises genomic DNA extraction reagent, probe quantitative PCR mix and positive and negative control, optionally genomic DNA extraction reagent is plant DNA extraction kit DP305-02 obtained from Tengen Biochemical Technology (Beijing) company limited, probe quantitative PCR mix is obtained from reagent kit RR391S from Bio-Engineering (Dalian) company limited, positive control is DNA of the transgenic soybean DAS44406 or plasmid DNA of the strain-specific sequence containing the transgenic soybean DAS44406 and the negative control does not contain a strain-specific sequence of transgenic soybean DAS44406. Preferred Method: The method involves (a) extracting DNA from the biological sample using DNA extraction reagent, amplifying the DNA obtained from the biological sample, as template using primer (A1), primer (A3), mix (M2) and mix (M1) by double PCR reaction, where PCR reaction is carried out at 94 degrees C for 5 minutes, 94 degrees C for 30 seconds, 60 degrees C for 30 seconds, 72 degrees C for 90 seconds and at 72 degrees C for 5 minutes for 15 cycle and amplifying the obtained PCR product, as template using primer (A2), primer (A4), probe (B1), probe (B2) and probe quantitative PCR mix by fluorescence PCR reaction, where PCR reaction is carried out at 95 degrees C for 1 minute, at 95 degrees C for 15 seconds and at 60 degrees C for 1 minute for 40 cycles and (b) determining whether the biological sample contains a strain-specific sequence of transgenic soybean DAS44406 based on the results of double nested fluorescence PCR amplification.
主权项：引物和探针组合，其特征在于，包括：引物一、引物二和探针一的组合；和引物三、引物四和探针二的组合；所述引物一由SEQ?ID?NO:1和SEQ?ID?NO:2组成，所述引物二由SEQ?ID?NO:3和SEQ?ID?NO:4组成，所述探针一为SEQ?ID?NO:5，所述引物三由SEQ?ID?NO:6和SEQ?ID?NO:7组成，所述引物四由SEQ?ID?NO:8和SEQ?ID?NO:9组成，所述探针二为SEQ?ID?NO:10。
申请号：CN201610915417.X
专利号：CN106282389-A
公开/公告号：CN106282389-A
申请/专利权人：中华人民共和国湛江出入境检验检疫局;中国检验检疫科学研究院
发明/设计人：袁俊杰;王莹;魏霜;马新华;龙阳;陈文;付伟;张娜;杨卓瑜;吴希阳
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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