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
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.
