Establishment Of A Fluorescent PCR Detection Method For Two Kinds Of Pathogens Of Infectious Diseases

Objective: infectious disease is a great human since the beginning of twenty-first Century,disease to human health and safety hazards.It has spread fast,infectious and harmful characteristics.Spread through respiratory and mosquito bites spread are the two main routes of transmission.As has caused worldwide concern(infectious atypical pneumonia SARS),avian influenza,H1N1 influenza,yellow fever and so on.Therefore,for rapid and accurate diagnosis of infectious virus is one of the key to protect the health and epidemic prevention and control of the public.However,traditional microbial testing now use most of the clinical health and medical institutions involved in training,separation and other steps,complicated operation,long detection period the purpose of this study is to establish a kind of detection spread through respiratory and mosquito bites spread two virus rapid and specific diagnostic method(PCR fluorescence probe).To avoid the general PCR Prone to false positive,cross contamination and other issues,so as to be able to carry out a rapid and accurate detection of the two viruses,and better guide clinical treatment.Methods: First of all,reading about human parainfluenza virus and Zika virus related literature,this study is to detect the specific gene,taking the opportunity and composite probe primer design basis,then to clinical samples as templates for preparing plasmid reference.Secondly,through the optimization of PCR reaction system and reaction conditions to determine the optimal concentration and proportion of the primers and probes.Finally,the evaluation of the sensitivity of the detection method,repeatability and specificity;sample selection of nucleic acid extraction reagent with the highest efficiency,the optimal amount of template;effect of different PCR instrument on the test results and clinical samples detection.Results:Literature to determine specific genes of human parainfluenza virus HN gene.The PCR reaction of the reaction system and the reaction conditions are determined,including HPIV1 and HPIV3,the final concentration in the downstream primer system was 0.4 mol/L probe in the system in the final concentration of 0.2 mol/L and HPIV2;the internal standard on the downstream primer final concentration in the system is 0.15 mol/L,the final concentration of probe is 0.06 mol/L;the HPIV1,HPIV2 and HPIV3 fluorescence probe and quenching probe and probe the ratio of F: Q = 1: 2 annealing temperature is 58 ?,the annealing time for the performance of 30 s.the end of the kit Kit showed that the minimum detectable concentration of not less than 5 x 102 copies/?l samples,repeated 10 times the results show that the CV value is less than 5%,10 varieties of animal pathogens and no amplification detection in the kit,good specificity and the two kinds of samples.The effect of nucleic acid extraction reagent is considerable,the sample dosage is 3 L.,and the results of three different models show that the different models have little effect on the experimental results.To determine the Zika virus specific gene NS5.Primers and probes the performance evaluation results show that with the primers and probes design principles.Detection sensitivity can reach to 5 x 102 copies/?l,duplicate detection results showed that the CV value is less than 10%.The establishment of the detection method and the other is non cross.Said the kit has good sensitivity,precision and specificity.Conclusion: This study successfully established a simultaneous detection of the human parainfluenza virus 3 subtypes and walled card virus diagnostic methods(PCR fluorescence probe method.The method has the advantages of high sensitivity,good repeatability,good specificity.For human parainfluenza virus and the walled card virus provides a specific,safe,fast,accurate detection method can used for the early diagnosis of the disease,to guide timely,accurately to take prevention and treatment measures.