A New Method Of Extraction With Elution-free Magnetic Beads And Its Application In Isothermal Amplification

Various diseases represented by COVID-19 pose a serious threat to human health.The realization of rapid and sensitive detection of various pathogenic microorganisms is the key link for effective prevention and control of epidemic diseases.Nucleic acid amplification tests(NAATs)has become one of the indispensable methods for the detection of pathogenic microorganisms.Obtaining high-quality nucleic acid extracts from biological samples plays an important role in ensuring the accuracy of NAATs.In this study,a new method for no-elution magnetic bead extraction was established,and the effect of this extraction method applied to LAMP extraction and detection of pathogenic microorganisms was analyzed,aiming to achieve rapid and efficient detection of various pathogenic microorganisms.Sensitive detection provides a high-quality nucleic acid sample basis.The main research content and results are as follows:In the first part,a new multi-sample rapid magnetic bead(MBs)extraction method,elution-free magnetic bead-based method,was established.In this part,the influence of MBs on the downstream amplification system was first discussed,and finally loop-mediated isothermal amplification(LAMP)was selected as the downstream amplification detection system of this method.Secondly,the different conditions of the extraction method were optimized.Finally,the rapid magnetic bead extraction method without elution was successfully constructed.Compared with the commercial magnetic bead extraction kit,the extraction time is shortened by about 77%,and the whole extraction process can be completed in about 7min.In the second part,the effect of extraction and detection of pathogenic microorganisms by elution-free magnetic bead extraction and real-time fluorescent quantitative LAMP was analyzed.This paper mainly evaluates the feasibility,specificity and sensitivity of fluorescent LAMP combined with no-elution magnetic bead extraction method for detecting different biological samples,and shows the effect of this method more intuitively by comparing with the fluorescent LAMP detection combined with commercial magnetic bead extraction kit.The no-elution magnetic bead extraction method combined with fluorescent LAMP can specifically detect different biological samples.The rapid elution-free magnetic bead extraction method can make the detection limit of fluorescence LAMP detection for RNA template reach 1.0×10~2copies/m L.The detection limit of DNA template is 1.0×10~2to 1.0×10~3CFU/m L,respectively.The sensitivity of fluorescence LAMP combined with this elution-free rapid extraction method based on magnetic beads is 10-1000 times higher than that of LAMP combined with commercial magnetic beads.In the third part,it is verified that the non-eluting magnetic bead-based method can be used for colorimetric LAMP detection.The sensitivity of non-eluting magnetic bead-based method for colorimetric LAMP detection of pathogenic microorganisms was analyzed and compared with commercial magnetic bead extraction kits.The results demonstrate that the target(the nucleic acid/MB composite)of the no-elution magnetic bead extraction method can be used for colorimetric LAMP detection.The detection limit of this method for RNA template is 1.0×10~3copies/m L,and the detection limit for DNA template is 1.0×10~3to 1.0×10~4CFU/m L,which is 100 times higher than the method of colorimetric LAMP combined with commercial magnetic bead extraction.In conclusion,the fast elution-free magnetic beads extraction method applied to LAMP detection can specifically and sensitively detect target pathogenic microorganisms from different biological samples.Similar to traditional magnetic bead extraction methods,the non-eluting magnetic bead extraction method also has the potential to be integrated into automatic nucleic acid detection machines.it makes it particularly suitable for the establishment of ultra-high-speed and sensitive analysis platforms for the detection of pathogens in various biological samples in centralized laboratories and remote sites.