Establishment of rapid detection of mycoplasma pneumoniae on the basis of multiple cross displacement amplification and nanoparticle-based biosensor
Objective To establish a simple,sensitive and rapid detection method for Mycoplasma pneumoniae(M.pneumoniae)and evaluate its application.Methods Multiple cross-displacement amplification(MCDA)re-action was used to amplify the CARDS toxin gene specific for M.pneumoniae,and lateral flow biosensor(LFB)technique was used to interpret the amplicons,and the method was termed as MP-MCDA-LFB.Assays performed at 60-67 ℃ were analyzed to obtain the optimal reaction temperature,and amplicons within different reaction time(10-40 min,10 min interval)were readout to obtain the optimal reaction time for MP-MCDA-LFB as-say.Serial dilutions of genomic DNA of M.pneumoniae were prepared for analytical sensitivity analysis and 35 non-M.pneumoniae strains were selected for analytical specificity analysis.Moreover,the MP-MCDA-LFB meth-od was further utilized to diagnose 80 clinical samples,then the obtained results were compared with that by RT-PCR method to validate the clinical feasibility of the new method.Results The MP-MCDA-LFB method was con-firmed feasible for quick diagnosis of M.pneumoniae.The optimum reaction temperature was 63 ℃,and the op-timal reaction time was 40 min,enabling the whole detection completed within 1 h.The MP-MCDA-LFB assay was confirmed highly sensitive and specific for M.pneumoniae detection,with a limit of detection at 45 ng/L and a specificity of 100％,displaying no cross-reaction with non-M.pneumoniae strains.In addition,the MP-MC-DA-LFB assay detected 45 positive samples from the 80 clinical samples(56.3％),which was completely in ac-cordance with that of RT-PCR assay.Conclusion The MP-MCDA-LFB detection method targeting CARDS toxin gene established in this study has the characteristics of simple,rapid,highly sensitive and strongly specific,ex-hibiting preferable potential of application in primary medical institutions and point-of-care tests.
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