title: [Construction of quantitative real-time PCR detection system of transgenic tomato line Zeneca B,Da,F].
abstract: OBJECTIVE
  To construct the plasmid reference molecules for detection of transgenic tomato line Zeneca B,Da,F using quantitative real-time PCR(qPCR).
  
  
  METHODS
  Three plasmid reference molecules pEasy-T3-APX, pEasy-T3-16A and pEasy-T3-16S were cloned based on reverse genetics, which contain the target fragments of tomato endogenous reference gene apx (ERG-apx), gene-specific sequence of pg(GS-pg) and construct-specific sequence of vectors pJR16S/pJR16A (CS-16S/CS-16A) of Zeneca B,Da,F, respectively. Primers and Taqman probes were designed by Beacon Designer 7.5.The specificity, sensitivity, reproducibility and the limit of detection(LOD) of the qualitative and quantitative PCR system based on the plasmid reference molecules were evaluated. PicoGreen was used to measure the DNA concentration of the plasmid reference molecules. Two sets of samples containing 1% or 0.1% (w/w) pEasy-T3-16A or pEasy-T3-16S mixed with pEasy-T3- APX as background DNA were prepared for evaluating the efficacy of the qPCR system.
  
  
  RESULTS
  The target fragments for qPCR detection were anchored, ERG-apx 108 bp, GS-pg 108 bp , CS-16S 109 bp and CS-16A 102 bp. The three plasmid reference molecules were confirmed at the expected sizes by restriction enzyme digestion. The qPCR results showed that the RSD of reproducibility were 0.2% to1.5%, LOD was 25 copies, R2 values for these standard curves were 0.994 ~0.998 and amplification efficiencies were 93.3%~102.4%.The bias between the test and true values of two sets of mixed samples ranged from -9.3% to 14.7% after adjusting by conversion factors(Cf).
  
  
  CONCLUSIONS
  The plasmid reference molecules and qPCR system for qualitative and quantitative detection of transgenic tomato line Zeneca B,Da,F have been established successfully.
authors.affiliation: Department of Parasitology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China.
authors.forename: Rong-Jia
authors.initials: RJ
authors.lastname: Mai
authors.forename: Min-Fang
authors.initials: MF
authors.lastname: Chen
authors.forename: Qian-Zhen
authors.initials: QZ
authors.lastname: Mo
authors.forename: Liang-Yong
authors.initials: LY
authors.lastname: Hu
authors.forename: Shi-Lun
authors.initials: SL
authors.lastname: Li
authors.forename: Min-Ran
authors.initials: MR
authors.lastname: Tang
authors.forename: Xiao-Min
authors.initials: XM
authors.lastname: Gu
authors.forename: Yong-Hong
authors.initials: YH
authors.lastname: Cai
authors.forename: Lun-Bin
authors.initials: LB
authors.lastname: Zhou
authors.forename: Xiao-Hong
authors.initials: XH
authors.lastname: Zhou
mesh_terms: D001483:Base Sequence; D018744:DNA, Plant / Q000235:genetics; D018551:Solanum lycopersicum / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics*; D010957:Plasmids; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D018744:DNA, Plant