中文摘要：本发明提供了一种转基因水稻Bt63的微滴式数字PCR检测方法及试剂盒。所述方法包括以下步骤提供了目标品系和内源基因的特异性引物和探针，样品的DNA提取，数字微滴式PCR扩增及结果的分析。本发明能精准定量的检测出米或米制品中是否含有转基因水稻Bt63，在各口岸转基因检测项目上实现了现场快速定量检测，具有很好的应用价值。
外文摘要：NOVELTY - Method for detecting transgenic rice Bt63 by droplet digital PCR, involves (a) obtaining primer and probe, preferably endogenous primers and probes for insect-resistant transgenic rice Bt63 and specific primers and probes for Bt63 lines of insect-resistant transgenic rice comprising Bt63-forward primer, (b) extracting to-be-detected sample, positive control and negative control DNA, (c) amplifying the DNA by performing droplet digital PCR amplification, and obtaining reaction liquid, and (d) detecting and analyzing the reaction liquid.
USE - The method or kit is useful for detecting transgenic rice Bt63 by droplet digital PCR (all claimed).
ADVANTAGE - The method accurately and quantitatively detects whether the rice or rice product contains the transgenic rice Bt63, and realizes rapid quantitativedetection on the site transgenic detection process.
DETAILED DESCRIPTION - Method for detecting transgenic rice Bt63 by droplet digital PCR, involves (a) obtaining primer and probe, preferably endogenous primers and probes for insect-resistant transgenic rice Bt63 comprising PLD-forward primer comprising base pair sequence of SEQ ID NO: 1, PLD-reverse primer comprising base pair sequence of SEQ ID NO: 2 and PLD-probe comprising base pair sequence of SEQ ID NO: 3 in which 5' end is labeled with FAM (RTM: 5-Carboxyfluorescein) and 3' end is labeled with TAMRA (RTM: 6-Carboxytetramethylrhodamine), and specific primers and probes for Bt63 lines of insect-resistant transgenic rice comprising Bt63-forward primer comprising base pair sequence of SEQ ID NO: 4, Bt63-reverse primer comprising base pair sequence of SEQ ID NO: 5 and Bt63-probe comprising base pair sequence of SEQ ID NO: 6 in which 5' end is labeled with FAM (RTM: 5-Carboxyfluorescein) and 3' end is labeled with Black hole quencher(BHQ)1 (RTM: Dye having broad absorption spanning of 480-580 nm), (b) extracting to-be-detected sample, posi! tive con trol and negative control DNA, (c) amplifying the DNA by performing droplet digital PCR amplification, and obtaining reaction liquid, and (d) detecting and analyzing the reaction liquid. tggtgagcgttttgcagtc (SEQ ID NO: 1), ctgatccactagcaggaggtc (SEQ ID NO: 2), tgttgtgctgccaatgtggcct (SEQ ID NO: 3), agagactggtgatttcagcgg (SEQ ID NO: 4), gcgtccagaaggaaaaggaat (SEQ ID NO: 5), and atctgccccagcactcgtcc (SEQ ID NO: 6). An INDEPENDENT CLAIM is included for kit, which comprises the above-mentioned primer and probe.
主权项：转基因水稻Bt63的微滴式数字PCR检测方法，包括以下步骤：(1)获得引物和探针，所述引物和探针包括：抗虫转基因水稻Bt63的内源引物和探针：PLD?Forward：TGGTGAGCGTTTTGCAGTCPLD?Reverse：CTGATCCACTAGCAGGAGGTCPLD?Probe：FAM?TGTTGTGCTGCCAATGTGGCCT?TAMRA抗虫转基因水稻Bt63品系的特异性引物和探针：Bt63?Forward：AGAGACTGGTGATTTCAGCGGBt63?Reverse：GCGTCCAGAAGGAAAAGGAATBt63?Probe：FAM?ATCTGCCCCAGCACTCGTCC?BHQ1；(2)提取待测样品、阳性对照、阴性对照的DNA；(3)将步骤(2)所得DNA进行数字微滴式PCR扩增，获得反应液；(4)对步骤(3)反应液进行检测与分析。
申请号：CN201611024354.5
专利号：CN106636360-A
公开/公告号：CN106636360-A
申请日：2016-11-18
公开/公告日：2017-05-10
申请/专利权人：浙江省检验检疫科学技术研究院
发明/设计人：张明哲 ; 张晓峰 ; 尹文秀 ; 徐莉莉 ; 吴姗
分类号：C12Q1/68
主分类号：C12Q1/68
法律状态：实质审查
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