| Background and objectivePathogen nucleic acid detection plays an important role in clinical diagnosis and treatment.Traditional pathogen culture requires a long time and complex operations,and nucleic acid based detection methods are more conducive to providing a variety of infection information.For example,human papilloma virus(HPV)gene detection is essential for cervical cancer screening.There are more than 200 HPV subtypes in the world,of which about 15 high-risk HPV subtypes can cause cancer.It is very important to establish real-time nucleic acid detection that can identify HPV subtypes in clinical samples for disease monitoring and treatment.Polymerase chain reaction(PCR)is the most commonly used pathogen nucleic acid detection method,but its practicality is limited by expensive equipment,cumbersome operation and high site requirements.Although many isothermal amplification methods without specific equipment have been reported,these methods have their own shortcomings.For example,loop mediated isothermal amplification(LAMP)has high requirements for primer design and operators,and It is easy to produce false-positive results.Therefore,it is urgent to develop a simple,sensitive and specific pathogen nucleic acid detection method.Methods1.Construction and characterization of a fluorescence detection method based on toe-hold mediated chain displacement initiated primer exchange reaction1.1 Design and synthesize various primer sequences required for primer exchange reaction.1.2 The assembled products and amplified products of various primer sequences were characterized by electrophoresis and fluorescence spectroscopy to verify the feasibility of this detection method.1.3 The one-step test method is adopted to verify the feasibility of one-step test.1.4 Optimize the reaction temperature,reaction time,primer length,DNA polymerase concentration,and obtain the best reaction conditions according to the experimental results.2.Evaluate the performance and clinical application ability of the constructed fluorescence platform2.1 Detect the fluorescence signals generated by the targets of different concentrations under the optimal reaction conditions,calculate the linear range and detection limit,and evaluate the analytical sensitivity of the method.2.2 The specificity of this method was evaluated by detecting the homologous DNA of multiple targets.2.3 Compare the detection efficiency of detection systems for HPV-16 targets at three different sites,and verify the importance of target selection.2.4 This method was used to detect DNA extracted by the kit and pyrolytic samples at the same time to evaluate the applicability of this method to pyrolytic methods.2.5This method was used to detect clinical samples,and compared with PCR method to evaluate the clinical detection performance of this method.ResultCompared with the traditional polymerase chain reaction and isothermal amplification,this method is simpler and faster.The detection specificity is improved by introducing a foothold mediated chain replacement reaction,and the powerful signal amplification ability of primer exchange reaction(PER)is combined to achieve high specificity and sensitivity detection.After optimization,the concentration of DNA polymerase in the reaction system is 0.2 U/μL and the length of Primer is 10 nt,which can reach the detection limit of 18 fM within 1 hour at a constant temperature of 37℃.At the same time,it is applicable to pyrolysis samples,further reducing the operation steps and detection time.The clinical application ability of the system was verified by 24 cervical exfoliated cell samples,30 plasma samples and 10 urine samples.This method is simple in operation,low in cost,widely applicable,and easy to integrate with the mature equipment on the market at present to realize POCT analysis of pathogen nucleic acids. |
PDF Full Text Request