Diseases caused by pathogen infections pose a serious threat to human health,and thus the technologies of pathogen detec-tion are topics of great concern to researchers.However,current pathogen identification methods have several problems such as the long time required to obtain test reports,high detection instrument costs,and large spatial requirements,which greatly limit the prevention and control of pathogens in resource-limited settings.Thus,a fast,accurate,low-cost and small space requirement detection method is of great significance in pathogen prevention and control.In this study,the magnetic beads method was employed for DNA extraction and purification,followed by the amplification of the target sequence using the recombinase polymerase amplification(RPA)assay.Subsequently,difference in fluorescence color was employed for visual judgment.The entire experimental process was designed on a microfluidic chip and applied for the rapid detection of important pathogens such as Klebsiella pneumoniae,Acinetobacter baumannii,Pseudomonas aeruginosa and Staphylococcus aureus.The results showed that DNA extraction and purification were completed within 40 min using the optimized microfluidic-magnetic beads method.Compared to other nucleic acid extraction methods,the optimized method provided higher concentration,better DNA purity and integrity,which could meet the needs of subsequent molecular biology ex-periments such as PCR;The rapid amplifications of species-specific genes were accomplished by a microfluidic-RPA method at a con-stant temperature(39 ℃)within 20 min.The universality of the method was validated by testing of 18 clinical isolates belonging to dif-ferent species in this study.There was a significant difference between the RPA products of the control and samples in fluorescence color,and the accuracy of the visual judgment results was further verified by sequencing.In conclusion,compared to traditional bench-top protocols,the microfluidic-based magnetic beads and RPA method for pathogen detection significantly shortened the time of detec-tion,made up for the shortcomings of traditional detection methods,and it has advantages such as lower equipment requirement,smal-ler space requirements and better integration,which may provide a fast,low-cost and simple supplementary solution for pathogen detec-tion.
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