[Objective] The aim was to construct a plasmid reference molecule (PRM) for detection of transgenic soybean MON89788. [Method] the lectin gene sequence,3'-junction and 5'-junction sequence between host plant D...[Objective] The aim was to construct a plasmid reference molecule (PRM) for detection of transgenic soybean MON89788. [Method] the lectin gene sequence,3'-junction and 5'-junction sequence between host plant DNA integrated DNA of MON89788 soybean were amplified independently,and the three fragments were cloned into the cloning vector pMD18-T in order through molecular manipulation method to construct pMD-LM3M5,the applicability of the constructed novel PRM was tested. [Result] Sequencing confirmation result showed that the PRM was 3 700 bp in length,containing 1 029 bp of recombined DNA fragment. The limits of qualitative detection of the PRM were 10 copies. [Conclusion] The PRM constructed in this study was suitable for the identification of MON89788 event.展开更多
The main advantage of digital PCR(dPCR) is that it facilitates absolute quantification of the target without reference to the standard/calibration curve.Crystal droplet dPCR has a three-color staining detection functi...The main advantage of digital PCR(dPCR) is that it facilitates absolute quantification of the target without reference to the standard/calibration curve.Crystal droplet dPCR has a three-color staining detection function,which enables multiplex PCR reaction.In this study,this technique was used to establish triple dPCR detection for the genetically modified soybean MON87708 × MON89788 with stacked traits.Specific absolute quantitative detection was accomplished for the genomic DNA extracted from the homogenized seeds of GM stack MON87708× MON89788 soybean.Our results can serve as a reference for the absolute quantitative detection of stacked events of genetically modified crops.展开更多
[Objective] The aim was to establish an event-specific qualitative PCR method for transgenic soybean MON89788.[Method] Firstly,the 3′-junction sequence between host plant DNA and integrated DNA of transgenic MON89788...[Objective] The aim was to establish an event-specific qualitative PCR method for transgenic soybean MON89788.[Method] Firstly,the 3′-junction sequence between host plant DNA and integrated DNA of transgenic MON89788 soybean was isolated using thermal asymmetric interlaced-PCR （TAIL-PCR）,and the specific PCR primers were designed based on the 3′-junction sequence.Secondly,the specificity and sensitivity of the qualitative PCR detection methods employing these primers were tested.[Result] 1 142-bp 3′-junction sequence was obtained.According to the sequence,event-specific qualitative PCR method was established,amplifying a 170-bp product specifically from MON89788 event,and the limit of detection was 0.05%,approximately 40 initial template copies.[Conclusion] The method was highly specific,sensitive,and suitable for detection of MON89788 event.展开更多
基金Supported by Major Projects of Cultivating New Varieties by Trans-genic Technology (2008ZX08012-001)~~
文摘[Objective] The aim was to construct a plasmid reference molecule (PRM) for detection of transgenic soybean MON89788. [Method] the lectin gene sequence,3'-junction and 5'-junction sequence between host plant DNA integrated DNA of MON89788 soybean were amplified independently,and the three fragments were cloned into the cloning vector pMD18-T in order through molecular manipulation method to construct pMD-LM3M5,the applicability of the constructed novel PRM was tested. [Result] Sequencing confirmation result showed that the PRM was 3 700 bp in length,containing 1 029 bp of recombined DNA fragment. The limits of qualitative detection of the PRM were 10 copies. [Conclusion] The PRM constructed in this study was suitable for the identification of MON89788 event.
文摘The main advantage of digital PCR(dPCR) is that it facilitates absolute quantification of the target without reference to the standard/calibration curve.Crystal droplet dPCR has a three-color staining detection function,which enables multiplex PCR reaction.In this study,this technique was used to establish triple dPCR detection for the genetically modified soybean MON87708 × MON89788 with stacked traits.Specific absolute quantitative detection was accomplished for the genomic DNA extracted from the homogenized seeds of GM stack MON87708× MON89788 soybean.Our results can serve as a reference for the absolute quantitative detection of stacked events of genetically modified crops.
基金Supported by Important National Science & Technology Specific Pro-jects(2008ZX08012-001)~~
文摘[Objective] The aim was to establish an event-specific qualitative PCR method for transgenic soybean MON89788.[Method] Firstly,the 3′-junction sequence between host plant DNA and integrated DNA of transgenic MON89788 soybean was isolated using thermal asymmetric interlaced-PCR （TAIL-PCR）,and the specific PCR primers were designed based on the 3′-junction sequence.Secondly,the specificity and sensitivity of the qualitative PCR detection methods employing these primers were tested.[Result] 1 142-bp 3′-junction sequence was obtained.According to the sequence,event-specific qualitative PCR method was established,amplifying a 170-bp product specifically from MON89788 event,and the limit of detection was 0.05%,approximately 40 initial template copies.[Conclusion] The method was highly specific,sensitive,and suitable for detection of MON89788 event.
