Development And Application Of A Novel Molecular Method To Detect Mycoplasma Pneumoniae By Interspersed Repeat Sequence PCR

Introduction:To develop a convenient,rapid,sensitive and specific PCR assay—IRS-PCR for the detection of M.pneumoniae for early diagnosis,follow-up and recurrence of pneumonia.And then detect M.pneumoniae of atypical pneumonia patients in TianJin.Meanwhile, the antibodies of M.pneumoniae and respiratory viruses were detected,and the results of respiratory viruses were collected in order to compare the differences of various pathogen.Methods:1.Culture and detection the concentration of Mac and clinical strains 1836,4299.2.17 pairs of IRS-PCR primers were designed at RepMP2/3 which were interspersed repeat sequence of M.pneumoniae with touchdown procedures.Detection of standard and clinical strain,sensitive and specific assays were included in primer screening.The sensitivity of the PCR was determined by using a viable suspension of Mac.Tenfold serial dilutions were made in culture medium to 10-10.Additional mycoplasma,respiratory organisms,and other common bacteria were tested to determine the specificity of the PCR,including E. coli, U. urealyticum, C. trachomatis, S.pneumoniae,K.pneumoniae, S.aureus, S. epid ermidis,StaphylococcusA,StaphylococcusB,P.aeruginosa,N.meningitidis,M.genita lium.After screening, we chose the best pair of primers for IRS-PCR.And then,the experiment was further optimized by L16 (45)orthogonal assay,which was used to determined the best combination of the most significant factors (primer,dNTP,TaqDNA polymerase,template and Mg2+)at their best levels,and performed in triplicate.Besides,we conducted additional experiments using the optimum IRS-PCR.3.IRS-PCR which was established and optimized above was applied in M.pneumoniae detection in 150 clinical samples of BALF.Moreover, the antibodies of M. pneumoniae were detected and respiratory viruses were collected.Finally,all the results were compared. Results:1.Standard and clinical strains:The color of liquid culture medium changed 3 to 5 days later."fried egg"colonies were shown on solid culture medium.The concentrations of Mac,1836 and 4299 were 107,108 and 107 by CCU,respectively.And the concentrations of their CFU were all 106/ml.2.Development and optimization of IRS-PCR:①F6R4 F3R2 and F4R4 were chosen by detection of standard and clinical strains.Their sensitivity were 10-9,10-8 and 10-8 CFU,respectively.F6R4 could amplify U.urealyticum,S.pneumoniae, StaphylococcusA,P.aeruginosa, N.meningitidis,andM.genitalium. F4R4 could amplify E.coli, M.genitalium.However, F3R2 was better than the other two primer pairs in specificity with good sensitivity.Thus, F3R2 were applied in IRS-PCR as primers.②All five factors had significant effect on IRS-PCR(P<0.05). Besides,The best combination of five factors were 0.6umol/L F3R2,0.3mmol/L dNTP,lU/25ul Taq DNA polymerase,2CFU DNA and 2.5mmol/L Mg2+. Statistics were analyzed by SPSS11.5.3.The positive rate of 150 samples by IRS-PCR was 41%.15(10%)positive cases of IgG were determined by serum antibody method.3 cases of RTV,2 cases of RSV and 1 case of AND were detected in clinical samples.The positve rate of M. pneumoniae was significantly higher than other respiratory pathogen(P<0.05).Conclusion:1.In conclusion,this report describes IRS-PCR that provides rapid and sensitive detection of M. pneumoniae, especially used in a large scale.Because of touchdown procedure,it's more specific than normal one.Therefore,it could be recommended for being wide application in early diagnosis of pneumonia and monitoring of recurrence.2.The positive rate of M. pneumoniae by IRS-PCR is significantly higher than other respiratory pathogen,and therefore M. pneumoniae infection should be pay more attention to,and could be applied in directing clinical drug usage.