Objective To construct reference standards for detection and quantification of Klebsiella pneumoniae(K.pneumoniae)with SYBR Green I-based real-time PCR assay.Methods Primers were designed based on the published sequen...Objective To construct reference standards for detection and quantification of Klebsiella pneumoniae(K.pneumoniae)with SYBR Green I-based real-time PCR assay.Methods Primers were designed based on the published sequence of the phoE gene of K.pneumoniae.The standard was prepared by cell culture,PCR and T-A clone methods,and was identified by colony PCR and DNA sequencing.Results The standard curve showed a very good linear negative regression between threshold cycle(Ct)and Log starting quantity of copy number.The detection range was from 5.2 to 5.2×106 copies per reaction,and the detection limit was 6 copies per reaction.The coefficients of variance(CVs)of three parallel experiments were in the range of 0.05%-0.91%.Conclusion The reference standards have high stability and reproducibility.They can be used in the quantitative detection of K.pneumoniae.展开更多
A plasmid containing target DNA and a standard curve for real-time quantitative PCR of the cold-induced Cor3 gene of Volvariella volvacea were constructed. These will provide the basis for further research on Cor3 gen...A plasmid containing target DNA and a standard curve for real-time quantitative PCR of the cold-induced Cor3 gene of Volvariella volvacea were constructed. These will provide the basis for further research on Cor3 gene expression at low temperature, and ultimately for assigning a role for the gene in the low temperature autolysis of V. volvacea.展开更多
转基因玉米MON87411是孟山都远东有限公司研发的抗虫耐除草剂玉米转化体,该转化体已获得进口加工原料的农业转基因生物安全证书。以转基因玉米MON87411品系特异性序列为靶标设计引物和探针,经特异性测试、体系优化、灵敏度测试及检出限...转基因玉米MON87411是孟山都远东有限公司研发的抗虫耐除草剂玉米转化体,该转化体已获得进口加工原料的农业转基因生物安全证书。以转基因玉米MON87411品系特异性序列为靶标设计引物和探针,经特异性测试、体系优化、灵敏度测试及检出限测试,建立了转基因玉米MON87411的实时荧光PCR定性检测方法。结果表明,该方法能检测出转基因玉米MON87411的转化体成分,检出限(limit of detection,LOD)可达0.05%,具有稳定性好、特异性强、灵敏度高的特点。国内8家转基因生物安全检测机构对本方法进行了特异性测试、检出限测试和再现性测试,循环验证报告显示该方法符合国家标准方法的各项要求,可在检测行业推广应用。研究建立的方法可为我国对转基因玉米MON87411品系的安全监管提供有效的技术支撑。展开更多
基金supported by the National High Technology Research and Development Program of China(863Program,No.2006AA06Z408)
文摘Objective To construct reference standards for detection and quantification of Klebsiella pneumoniae(K.pneumoniae)with SYBR Green I-based real-time PCR assay.Methods Primers were designed based on the published sequence of the phoE gene of K.pneumoniae.The standard was prepared by cell culture,PCR and T-A clone methods,and was identified by colony PCR and DNA sequencing.Results The standard curve showed a very good linear negative regression between threshold cycle(Ct)and Log starting quantity of copy number.The detection range was from 5.2 to 5.2×106 copies per reaction,and the detection limit was 6 copies per reaction.The coefficients of variance(CVs)of three parallel experiments were in the range of 0.05%-0.91%.Conclusion The reference standards have high stability and reproducibility.They can be used in the quantitative detection of K.pneumoniae.
基金Sponsored by the Shanghai Academy of Agricultural Science and Shanghai Science Committee (No .003912076)
文摘A plasmid containing target DNA and a standard curve for real-time quantitative PCR of the cold-induced Cor3 gene of Volvariella volvacea were constructed. These will provide the basis for further research on Cor3 gene expression at low temperature, and ultimately for assigning a role for the gene in the low temperature autolysis of V. volvacea.
文摘转基因玉米MON87411是孟山都远东有限公司研发的抗虫耐除草剂玉米转化体,该转化体已获得进口加工原料的农业转基因生物安全证书。以转基因玉米MON87411品系特异性序列为靶标设计引物和探针,经特异性测试、体系优化、灵敏度测试及检出限测试,建立了转基因玉米MON87411的实时荧光PCR定性检测方法。结果表明,该方法能检测出转基因玉米MON87411的转化体成分,检出限(limit of detection,LOD)可达0.05%,具有稳定性好、特异性强、灵敏度高的特点。国内8家转基因生物安全检测机构对本方法进行了特异性测试、检出限测试和再现性测试,循环验证报告显示该方法符合国家标准方法的各项要求,可在检测行业推广应用。研究建立的方法可为我国对转基因玉米MON87411品系的安全监管提供有效的技术支撑。
