Traditional transgenic detection methods require high test conditions and struggle to be both sensitive and efficient.In this study,a one-tube dual recombinase polymerase amplification(RPA)reaction system for CP4-EPSP...Traditional transgenic detection methods require high test conditions and struggle to be both sensitive and efficient.In this study,a one-tube dual recombinase polymerase amplification(RPA)reaction system for CP4-EPSPS and Cry1Ab/Ac was proposed and combined with a lateral flow immunochromatographic assay,named“Dual-RPA-LFD”,to visualize the dual detection of genetically modified(GM)crops.In which,the herbicide tolerance gene CP4-EPSPS and the insect resistance gene Cry1Ab/Ac were selected as targets taking into account the current status of the most widespread application of insect resistance and herbicide tolerance traits and their stacked traits.Gradient diluted plasmids,transgenic standards,and actual samples were used as templates to conduct sensitivity,specificity,and practicality assays,respectively.The constructed method achieved the visual detection of plasmid at levels as low as 100 copies,demonstrating its high sensitivity.In addition,good applicability to transgenic samples was observed,with no cross-interference between two test lines and no influence from other genes.In conclusion,this strategy achieved the expected purpose of simultaneous detection of the two popular targets in GM crops within 20 min at 37°C in a rapid,equipmentfree field manner,providing a new alternative for rapid screening for transgenic assays in the field.展开更多
CRISPR/Cas system,originally developed as genetic editing tool,also shows great potentials for nucleotide detection.A recent study published in Molecular Cell(Freije et al.,2019)developed a Cas13a-based CARVER(Cas13-a...CRISPR/Cas system,originally developed as genetic editing tool,also shows great potentials for nucleotide detection.A recent study published in Molecular Cell(Freije et al.,2019)developed a Cas13a-based CARVER(Cas13-assisted restriction of viral expression and readout)to detect RNA viruses such as lymphocytic choriomeningitis,influenza A and vesicular stomatitis,which provided a potential expanded application for the detection of a broad range of viral nucleotides in disease diagnosis.展开更多
基金supported by the Scientific and Innovative Action Plan of Shanghai(21N31900800)Shanghai Rising-Star Program(23QB1403500)+4 种基金the Shanghai Sailing Program(20YF1443000)Shanghai Science and Technology Commission,the Belt and Road Project(20310750500)Talent Project of SAAS(2023-2025)Runup Plan of SAAS(ZP22211)the SAAS Program for Excellent Research Team(2022(B-16))。
文摘Traditional transgenic detection methods require high test conditions and struggle to be both sensitive and efficient.In this study,a one-tube dual recombinase polymerase amplification(RPA)reaction system for CP4-EPSPS and Cry1Ab/Ac was proposed and combined with a lateral flow immunochromatographic assay,named“Dual-RPA-LFD”,to visualize the dual detection of genetically modified(GM)crops.In which,the herbicide tolerance gene CP4-EPSPS and the insect resistance gene Cry1Ab/Ac were selected as targets taking into account the current status of the most widespread application of insect resistance and herbicide tolerance traits and their stacked traits.Gradient diluted plasmids,transgenic standards,and actual samples were used as templates to conduct sensitivity,specificity,and practicality assays,respectively.The constructed method achieved the visual detection of plasmid at levels as low as 100 copies,demonstrating its high sensitivity.In addition,good applicability to transgenic samples was observed,with no cross-interference between two test lines and no influence from other genes.In conclusion,this strategy achieved the expected purpose of simultaneous detection of the two popular targets in GM crops within 20 min at 37°C in a rapid,equipmentfree field manner,providing a new alternative for rapid screening for transgenic assays in the field.
基金The work was supported by grants from the National Key Research and Development Program of China(Grant No.2017YFA0103902)the National Natural Science Foundation of China(Grant No.31771283)+3 种基金the Fundamental Research Funds for the Central Universities of Tongji University(Grant No.22120190210)the SAAS Program for Excellent Research Team(Grant No.2017-B-07)the Shanghai Leading Talent Project(Grant No.2017067)the Shanghai Fresh Corn Technology System Project(Grant No.2017-10).
文摘CRISPR/Cas system,originally developed as genetic editing tool,also shows great potentials for nucleotide detection.A recent study published in Molecular Cell(Freije et al.,2019)developed a Cas13a-based CARVER(Cas13-assisted restriction of viral expression and readout)to detect RNA viruses such as lymphocytic choriomeningitis,influenza A and vesicular stomatitis,which provided a potential expanded application for the detection of a broad range of viral nucleotides in disease diagnosis.
