Development And Clinical Application Study On A Detective Method Of EBV Gene By Ramification Amplification Method

This study set up ramification amplification method (RAM), which is a new method to detect EB virus gene. We have detected the EB virus gene in peripheral blood of patients of non-Hodgkin's lymphoma and nasopharyngeal carcinoma (NPC) patients by ramification amplification method (RAM) . RAM is utilizing a circular probe (C-probe) to finish gene amplification based on PCR. C-probe is a kind of nucleic acid probe with unique design. It includes three regions: the complementation sequence of 5', 3'end and target nucleic acid and the gene-linked area between 5'and 3'end . Once C-probe hybrid with target gene, its 5'and 3'end will close into circular shape acted by DNA ligase and then twist on the target gene with corkscrewing. When C-probe hybridizes to a target, a DNA polymerase extends the bound forwards primer along C-probe and continuously displaces a downstream strand, generating a multimeric single-stranded DNA(ssDNA).Then this ssDNA serve as a template for multiple reverse primers to hybridize, extend, and displace downstream DNA, generating a large ramified DNA complex, and resulting in an exponential amplification under an isothermal condition.Method:RAM method: Lysis the cells into 5M GTC solutions, 60℃ 1h, 37℃ overnight ( to make protein denaturation, inactivate nuclease and rarefacte the structure of double-strand DNA).Then mix the sample(or target gene) with C-probe, capture probe and hybridization buffer fully, 55℃ water bath 1h and then add magnetic beads. The biotin that combined with the capture probe will combine with the streptomycin avidin which located on the magnetic beads. Next catch the probe-target gene-circle probe complex through magnetic separation and use TE buffer to wash the magnetic beads so as to remove the unhybridizative probe and cells components.Then add Taq DNA ligase, 60℃ 20min, making the probe to be a circle by connecting the the 5'and 3'end , and combined the probe on the target gene, then add the RAM primer, Bst DNA polymerase, dNTP, T4 gene protein, DMSO, hybridization buffer, 63oC 1h, then inactivated Bst DNA ploymerase at 95 oC. All the product multiplied by RAM are cut by Ecol I at 37oC for one night, then analyzed the results by agarose gel(2%) electrophoresis.Testing the sensitivity of RAM:Chemical synthesized EBER-1 gene sequence were diluted into different concentration(108 107 106 105 104 103 102 101 ) as a target gene to test the sensitivity of RAM.PCR testing: total volume of the system is 50ul,the mixture of the response contains 5ul 10×PCR buffer, 300uMdNTP, 0.6pmol PCR upstream and downstream primer, 2u Tag DNA polymerase and 0.1ug sample DNA. The condition is 94℃5min,94℃ 1min,54℃ 1min,72℃ 1min for 35 circles. After the last circle, 72℃ 5min, 2% agarose gel electrophoresis.Hybridization in situ: Prepare the cells smear, detect the EBER-1 gene in the cells by the probe that marked by biotin and then operate according to the instruction of the kit Statistics method: Adopted the 田口玄一'accumulation analysis of variance to do the statistics analysis.Results: 1. RAM is a suitable and convenient method to use in basic hospital because of its high sensitivity, strong specificity and simple operation.RAM has hypersensitivity: RAM amplification products are increased by means of exponential, that is amplificating tiny DNA to the level that can be seen under the ultra voilet. It has been proved that RAM can detect 10 molecules target gene at least. This is equal to the sensitive of PCR. RAM has hyper-specificity: In RAM reaction, the formation of closed C-probe is based on C-probe combine with target DNA specific, so the specificity of the reaction can be improved greatly. On the other hand, using capture probe can also increase the specificity of the reaction because the target DNA can be caught onto magnetic beads after it hybridizate with capture probe through certain sequence and then a target DNA-C-probe- capture probe compound will form.The operation of RAM is convenient and rapid: The use of magn...