Preliminary Application Of Visual Method Based On G-quadruplex And Ultrasensitive HRCA Technology In Detection Of DNA

Objective: 1.Aim to provides a convenient and cheap method for clinical detection of cytomegalovirus a novle visual detection HCMV Nest PCR production with DNAzyme was developed.2.Aim to provides a convenient andsensitive method for clinical detection resistance gene of liver failure based on HRCA was developed.Methods: 1.A novle visual detection HCMV Nest PCR production with DNAzyme was developed: The target DNA undergoes the first run of polymerase chain reaction with the first set of primers,then the product from the first reaction undergoes a second run with the second set of primers.we design a stable sequence attached to the second set of forward primer,during amplication will produce a DNA strand have a universal tag complementary to the fixed sequence on the forward primers.Then an oligonucleotide probe was used.The probe is a hairpin structure will open and format G-quadruplex when it bind to universal tag.In the presence of heimn,formatting G-quadruplex-hemin complexes efficiently catalyzing the TMB,at last TMB turn colors,can observe by naked eye.Optimize the experimental conditions and evaluate the performance of the method.2.A novel detection resistance gene of liver failure based on HRCA was developed.For address those issues,in this study we designed two method for detecting target DNA,the second method was proved unworkable,the principle of second metod is that PLP will become a loop if the reaction solution have mutation gene,then the excision enzyme was added to reaction solution aim to Reduce the background.Sequently,the primers,SYBR Green I and DNA polymerase was added to reaction solution for turning on HRCA.Finally,through the change of the fluorescence intensity to determine the content of the target DNA.Optimize the experimental conditions and evaluate the performance of the method.Result: 1.A novel visual detection HCMV Nest PCR production with DNAzyme was developed.The experimental condition was optimized as follows:chose PCR buffer with 400 mm Na Cl and long universal tag for experiment,give up using asymmertric PCR for amplification,found that there is no different between G repetitive sequence on 5' or on 3',the sensitive was 82.1 compies/ml,intraassay CV=5.41%,inter assay CV=5.62%,which shows the methods have a good specificityand sensitivity There was no different between the results of real-time fluorescent quantitative PCR and the new mothed with detection for 20 positive samples.2.A novel detection resistance gene of liver failure based on HRCA was developed.The experimental condition was optimized as follows: chose the 3U E.coli;1u M PLP;1U Bst DNA polymerase for experiment,Draw the standard curve :Y=0.61X+508.11,R=0.99985,the sensitivity was0.05 nmol/L,and have good specificity.Intraassay CV=4.03%,inter assay CV=3.71%,it was means that the method have good repeatability.Conclusion: A novle visual detection HCMV Nest PCR production with DNAzyme was developed and A novel detection resistance gene of liver failure based on HRCA which have sensitivity,specificity,low-cost and convenient were been developed.,Then the experiment conditions were optimized for two method,which may beused in the application of clinical and laid the Foundation for Visual detectionn ucleic acid with isothermal amplification method.