中文摘要：本发明公开了一种转基因水稻多重数字PCR定量检测方法，属于转基因水稻的定量检测领域。本发明首先公开了用于检测转基因水稻KF2、KF6、KF8、KMD1、M12、TT51‑1和G6H1的多重数字PCR定量检测引物对和探针。在此基础上，本发明进一步建立了一种转基因水稻KF2、KF6、KF8、KMD1、M12、TT51‑1和G6H1的多重数字PCR定量检测方法。本发明方法能够实现对同时含有KF2、KF6、KF8、KMD1、M12、TT51‑1和G6H1 7种或其中几种转基因水稻的样品进行定量检测，而且对转基因成分定量检测的稳定性高，是一种水稻转基因成分定量检测的有效方法。
外文摘要：NOVELTY - Multiplex digital PCR quantitative detection primer pair and probe, is claimed. The transgenic rice KF2 transformant-specific primer pair comprises upstream sequence of SEQ ID NO: 1 and downstream sequence of SEQ ID NO: 2 and probe comprises base pair sequence of SEQ ID NO: 3. The transgenic rice KF6 transformant-specific primer pair comprises upstream sequence of SEQ ID NO: 4 and downstream sequence of SEQ ID NO: 5 and probe comprises base pair sequence of SEQ ID NO: 6.
USE - The multiplex digital PCR quantitative detection primer pair and probe in kit is useful for detecting transgenic rice KF2, KF6, KF8, KMD1, M12, TT51-1, and G6H1 (all claimed).
ADVANTAGE - The multiplex digital PCR quantitative detection primer pair and probe detects transgenic rice with high stability.
DETAILED DESCRIPTION - Multiplex digital PCR quantitative detection primer pair and probe comprises. The transgenic rice KF2 transformant-specific primer pair comprises upstream sequence of SEQ ID NO: 1 and downstream sequence of SEQ ID NO: 2 and probe comprises base pair sequence of SEQ ID NO: 3. The transgenic rice KF6 transformant-specific primer pair comprises upstream sequence of SEQ ID NO: 4 and downstream sequence of SEQ ID NO: 5 and probe comprises base pair sequence of SEQ ID NO: 6. The transgenic rice KF8 transformant-specific primer pair comprises upstream sequence of SEQ ID NO: 7 and downstream sequence of SEQ ID NO: 8 and probe comprises a 36 base pair sequence of (SEQ ID NO: 9) fully defined in the specification. The transgenic rice KMD1 transformant-specific primer pair comprises upstream sequence of SEQ ID NO: 10 and downstream sequence of SEQ ID NO: 11 and probe comprises base pair sequence of SEQ ID NO: 12. The transgenic rice M12 transformant-specific primer pair comprises upstream sequence of SEQ ID NO: 13 and downstream sequence of SEQ ID NO: 14 and probe comprises base pair sequence of SEQ ID NO: 15. The transgeni! c rice T T51-1 transformant-specific primer pair comprises upstream sequence of SEQ ID NO: 16 and downstream sequence of SEQ ID NO: 17 and probe comprises base pair sequence of SEQ ID NO: 18. The transgenic rice G6H1 transformant-specific primer pair comprises upstream sequence of SEQ ID NO: 19 and downstream sequence of SEQ ID NO: 20 and probe comprises base pair sequence of SEQ ID NO: 21. gctcgtctggctaagatcg (SEQ ID NO: 1), gcacggactatacaagttgtgat (SEQ ID NO: 2), cagcgatcgcatccttaggtcaaagcggtt (SEQ ID NO: 3), gcttggatcagattgtcgttt (SEQ ID NO: 4), gtcagataaactgattggtctgat (SEQ ID NO: 5), cgacaaaagatcaggatttggg (SEQ ID NO: 6), agtttcttaagattgaatggttgcc (SEQ ID NO: 7), cgaccatgatgctgttctgc (SEQ ID NO: 8), tggtgagcgttttgcagtct (SEQ ID NO: 10), ctgatccactagcaggaggt cc (SEQ ID NO: 11), tgttgtgctgccaatgtggcctg (SEQ ID NO: 12), caagtccttccagtattttgca (SEQ ID NO: 13), taccgcatcaggcgct (SEQ ID NO: 14), ctgatctctagtgctccagcgagtc (SEQ ID NO: 15), agagactggtgatttcagcggg (SEQ ID NO: 16), gcgtccagaaggaaaaggaata (SEQ ID NO: 17), atctgccccagcactcgtccg (SEQ ID NO: 18), aagcgtcaatttgtttacacc (SEQ ID NO: 19), tcgatctgctgcagcttg (SEQ ID NO: 20) and atggatgtatcgccaccagc acc (SEQ ID NO: 21). INDEPENDENT CLAIMS are included for the following: (1) method for performing quantitative detection of transgenic rice involves (a) extracting DNA of the rice sample to be detected, (b) using extracted DNA as template, and performing multiplex digital PCR amplification using rice internal reference gene detection primer pair and probe, and (c) performing data analysis of the amplification result, and calculating the ratio of the copy number of foreign gene and the copy number of the internal reference gene to obtain quantitative value of the transgenic component; and (2) kit, comprising ddPCR (RTM: Droplet digital PCR Supermix for probe), rice internal reference gene detection primer pair and probe, and multiplex digital PCR quantitative detection primer pair and probe, where the 5' end of DNA rice internal refe! rence pr obe is labeled with HEX (RTM: 2',4',5',7',1,4-Hexachlorofluorescein), and the 3' end is labeled with BHQ1 (RTM: Black hole quencher-1), and 5' end of multiplex digital PCR quantitative detection probe is labeled with FAM (RTM: 5-Carboxyfluorescein), and the 3' end is labeled with BHQ1 (RTM: Black hole quencher-1).
主权项：用于检测转基因水稻KF2、KF6、KF8、KMD1、M12、TT51-1和G6H1的多重数字PCR定量检测引物对和探针，其特征在于，包括：由核苷酸序列为SEQ ID No.1所示上游引物和SEQ ID No.2所示下游引物组成的转基因水稻KF2转化体特异性引物对；转基因水稻KF2转化体特异性探针的核苷酸序列为SEQ ID No.3所示；由核苷酸序列为SEQ ID No.4所示上游引物和SEQ ID No.5所示下游引物组成的转基因水稻KF6转化体特异性引物对；转基因水稻KF6转化体特异性探针的核苷酸序列为SEQ ID No.6所示；由核苷酸序列为SEQ ID No.7所示上游引物和SEQ ID No.8所示下游引物组成的转基因水稻KF8转化体特异性引物对；转基因水稻KF8转化体特异性探针的核苷酸序列为SEQ ID No.9所示；由核苷酸序列为SEQ ID No.10所示上游引物和SEQ ID No.11所示下游引物组成的转基因水稻KMD1转化体特异性引物对；转基因水稻KMD1转化体特异性探针的核苷酸序列为SEQ ID No.12所示；由核苷酸序列为SEQ ID No.13所示上游引物和SEQ ID No.14所示下游引物组成的转基因水稻M12转化体特异性引物对；转基因水稻M12转化体特异性探针的核苷酸序列为SEQ ID No.15所示；由核苷酸序列为SEQ ID No.16所示上游引物和SEQ ID No.17所示下游引物组成的转基因水稻TT51-1转化体特异性引物对；转基因水稻TT51-1转化体特异性探针的核苷酸序列为SEQ ID No.18所示；以及，由核苷酸序列为SEQ ID No.19所示上游引物和SEQ ID No.20所示下游引物组成的转基因水稻G6H1转化体特异性引物对；转基因水稻G6H1转化体特异性探针的核苷酸序列为SEQ ID No.21所示。
申请号：CN201710117885.7
专利号：CN106868137-A
公开/公告号：CN106868137A
申请/专利权人：浙江省农业科学院;
发明/设计人：汪小福; 徐俊锋; 陈笑芸; 彭城; 徐晓丽; 魏巍
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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