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
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.
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。
