A Novel Method For The Detection Of Mycoplasma Pneumoniae By Silica Membrane Solid Phase Extraction DNA And Isothermal Amplification

Community-acquired pneumonia(CAP)is the cause of morbidity and mortality in humans.Mycoplasma pneumoniae is the most frequent bacterial species leading to CAP.Thus,accurate diagnosis is urgently required for point-of-care testing(POCT)preventing and controlling the large-scale outbreak of M.pneumoniae-caused CAP.In order to detect M.pneumoniae quickly and accurately,an amino-modified silica membrane(AMSM)modified with N-[3-(Trimethoxysilyl)propyl] ethylenediamine(DAMO)was developed in this study.The membrane that we had proposed is mainly used for DNA extracting.Compared with the blank membrane,AMSM has an increased the adsorption capacity of DNA about 11 times.The adsorption capacity of AMSM with 3 mm diameter was about 133.5 ng,the adsorption capacity per unit area was about 18.89 ng/mm2,and the adsorption efficiency was nearly 90%.AMSM had a high DNA adsorption capacity and stable adsorption ability.Based on the AMSM,this study proposed a simple solid-phase DNA extraction method that does not need high-speed centrifugation steps,which can achieve rapid DNA capture and enrichment within 3 min.It provided a basis for the further development of new method for the detection of M.pneumoniae based on integrated solid-phase DNA extraction and nucleic acid amplification.Isothermal nucleic acid amplification technology doesn’t have the need of expensive equipment which made it can be extensively applied in the resource-limited area.And Isothermal nucleic acid amplification technology is the suitable candidate for POCT detecting M.pneumoniae.In this study,a rapid,simple,and sensitive method for detecting M.pneumoniae was proposed by combining the AMSM-based DNA extraction with isothermal amplification.AMSM didn’t have a significant inhibitory effect on the loop-mediated isothermal amplification(LAMP)reaction and the strand exchange amplification(SEA)reaction.The AMSM/DNA complexes can be directly transferred into isothermal amplification mixture without DNA elution step.According to the sensitivity experiment,samples containing 1.0 × 100 copies/μL of M.pneumoniae DNA could be detected by AMSM-based colorimetric LAMP,and samples containing 1.0 × 101 copies/μL of M.pneumoniae DNA could be detected by AMSM-based colorimetric SEA.In addition,45 real samples of M.pneumoniae were tested.The results showed that the positive coincidence rate of LAMP method combined with AMSM was 96.67%,and that of SEA method combined with AMSM was 93.33%.The AMSM-based detection protocol for M.pneumoniae proposed in this study can be completed within 60 min without high-speed centrifugation steps,and the detection results can be naked-eye visualized.In conclusion,the detection scheme of M.pneumoniae proposed in this study is simpler and faster than traditional methods.The method proposed in this study can be used for the detection of low-concentration samples and is suitable for the rapid detection of M.pneumoniae in resource-limited areas.It is also the ideal candidate of POCT.It provided a method reference for the future development of automated detection devices and the rapid detection of other pathogens which has broad application prospects.