Research On The Rapid Detection Method Of Mycoplasma Pneumoniae Nucleic Acid Based On The New Method Of Strand Exchange Amplification

Mycoplasma pneumoniae(M.pneumoniae)is one of the main pathogens causing respiratory infections and is a common cause of upper respiratory tract infections.With the deterioration of air quality in the environment in recent years,more and more people has suffer from respiratory infections,and the diagnosis of respiratory infections has gradually become the focus of attention.There is no difference between the respiratory tract infection caused by M.pneumoniae and the symptoms of common respiratory tract infection.However,if not diagnosed and treated in time,it may cause multiple organ damage and organic disease in the whole body to endanger the life.Therefore,it is very meaningful to develop an accurate and rapid method for detecting M.pneumoniae.In this study,M.pneumoniae was used as a research object,and the method of rapid detection of nucleic acid for M.pneumoniae was studied by Denaturation bubble-meditated strand exchange amplification(SEA).The main research contents and conclusions are as follows:1.Comparative study on nucleic acid extraction of M.pneumoniae in sputum samples:Sputum samples from 10 patients with M.pneumoniae infection,Phenol-chloroform nucleic acid extraction method,magnetic bead nucleic acid extraction method and centrifugal column nucleic acid extraction method were used to extract and determine nucleic acid from sputum after digestion of 300μL Sputum samples.The extraction efficiency of the three methods was compared to select an optimal method for nucleic acid extraction from sputum of M.pneumoniae infection.The extraction efficiency of the three methods was compared to select an optimal method for nucleic acid extraction from sputum of M.pneumoniae infection.The results showed that the concentration range of nucleic acid extracted by centrifugal column was 255.6-440.3 ng/μL,that by magnetic bead extraction was 110-186.9 ng/μL,and that by phenol-chloroform extraction was 11.6-27.5 ng/μL.The ratio of OD260/280 to OD260/230 was used to compare the purity of nucleic acid extracted by the three methods,the ratio of OD260/280 of the three nucleic acid extraction methods of spin column,magnetic beads and phenol-chloroform was 2.51-3.15,1.53-1.93 and 1.32-1.85.OD260/230 ratio ranges from 2.77-3.22,0.52-1.98 and 0.32-1.75 respectively.Conclusion:The concentration,extraction time and amplification efficiency of M.pneumoniae nucleic acid extracted from sputum were analyzed and compared by three methods.The centrifugal column nucleic acid extraction method was the best one among the three methods.2.Establishment of a new isothermal method based on strand exchange amplification for rapid detection of M.pneumoniae nucleic Acid:With M.pneumoniae as the target,a new method for detection of M.pneumoniae was established through the study of primer design,feasibility,reaction temperature optimization,sensitivity,specificity and visualization of reaction results of SEA method for detection of M.pneumoniae.The results showed that the lowest concentration of M.pneumoniae was 1×10~4 copy/mL at the optimum temperature of 61℃.The primers of M.pneumoniae detected by SEA had good specificity and could distinguish 11 main pathogenic bacteria causing upper respiratory tract infection.Conclusion:The new method of SEA isothermal amplification can detect the nucleic acid of M.pneumoniae quickly and specifically,and the reaction results are visualized.it does not need to rely fluorescent instrument to observe the results.3.SEA for the detection of actual samples and comparative studies of three methods:The sputum samples from 224 patients with upper respiratory tract infection were detected by SEA,Loop-mediated isothermal amplification(LAMP)and Polymerase chain reaction(PCR)respectively and the accuracy of the results of the three methods was compared.SEA,LAMP and PCR detection methods respectively detected 38,41and 42 positive samples of infection in 224 cases of sputum samples,but the LAMP method detected 2 false positive results in the actual sample test,the positive detection rates of the three methods were16.96%,18.30%,and 18.75%respectively.At the same time,the differences of the three methods in reaction threshold time,number of primers,detection limit and reaction temperature were analyzed.Conclusion:The accuracy of SEA detection of M.pneumoniae is slightly lower than that of PCR and LAMP.However,this method has its own advantages in primer number,reaction time and reaction conditions.