ABSTRACT
OBJECTIVE@#To establish a rapid method for EBV detection with loop-mediated isothermal amplification (LAMP), and to make it as a clue for early diagnosis of nasopharyngeal carcinoma cancer.@*METHOD@#EBV DNA was fast extracted from samples after boiling, while the whole detection will be finished within an hour with specific amplification of EBV gene by LAMP.@*RESULT@#High specificity was shown from EBV detection of 33 clinical samples. Comparing with PCR, LAMP is more simple and convenient to perform under isothermal conditions, and require no special apparatus, thus, it is more economical and practical.@*CONCLUSION@#LAMP analysis of EBV may be an efficient and easy way for clinical diagnosis of nasopharyngeal carcinoma.
Subject(s)
Humans , DNA Primers , DNA, Viral , Herpesvirus 4, Human , Genetics , Molecular Sequence Data , Nasopharyngeal Neoplasms , Diagnosis , Nucleic Acid Amplification Techniques , Sensitivity and SpecificityABSTRACT
Enhanced green fluorescent protein( EGFP), myc epitope and polyhistidine metal-binding tag are often used as a marker for recombinant fusion protein in many gene expression vectors, each marker has its own function, EGFP emits green fluorescence for direct detection, myc epitope facilitates recombinant fusion protein detection using its antibodies, polyhistidine tag allows purification of recombinant fusion protein using resin.Hitherto, no a plasmid vector can integrate all of these functions. In this study we constructed a novel eukaryotic expressive plasmid, designated as pcDNA6/myc-his-EGFP B, which integrated the functions of EGFP, myc epitope and polyhistidine tag. Importantly, a linker octo - peptide in N terminal of EGFP was designed using LINKER program. A DNA fragment encoding a putative protein containing a signal peptide of human interleukin 2(IL-2) in N terminal was cloned into pcDNA6/myc-his-EGFP B in frame with the C-terminal peptide to construct pMHES. 2.2.15 Cells were transfected with pcDNA6/myc-his-EGFP B and pMHES, and Balb/c mice were intravenously injected with pcDNA6/myc-his-EGFP B by tails, results revealed that both of the plasmids worked in 2.2.15 Cells and livers of Balb/c mice. Assuming gene of the IL-2 was inserted into pcDNA6/myc-his -EGFP B in frame with EGFP, myc and 6 × His, three-dimensional structure for this putative expression product was simulated using Modeller8V2, results revealed that IL-2, EGFP, myc and 6 × his did not interfere each other and octo- peptide linker owned certain flexibility. The results suggest that pcDNA6/myc-his-EGFP B may be useful as a genetic tool for mammalian cells and a vector for gene therapy.