Microfluidic Isothermal Amplification Technology For The Rapid And Field Detection Of Nucleic Acids Of Monkeypox And Related Virus

Objective:The epidemic situation of COVID-19 highlights the importance of prevention and control of new outbreaks of infectious diseases.In addition to COVID-19,the monkeypox virus outbreak has also become a public health emergency worthy of attention by the World Health Organization(WHO)because it first spilled over to Africa in 2022.Pandemic outbreaks,such as monkeypox(MPXV),Ebola(EBOV),Marburg(MARV),Rift Valley fever(RVFV),Lassa fever(LARV),yellow fever(YFV),and smallpox virus(VARV)in Africa,pose a serious threat to human health and social stability.the use of traditional detection methods for screening and confirmation of suspected sufferers is severely restricted due to the need for trained technicians,rather complicated instrumentation,and a biosafety experiment cabinet.It is urgent to develop the detection technology applicable to the field.Loop-mediated isothermal amplification(LAMP)has received widespread attention for its ability to specifically detect target nucleic acids in as few as a few copies over 30-60 min using 4-6 primer sets under constant temperature conditions;Microfluidic technology has the advantages of integrated miniaturization,automation,and high throughput.Therefore,in this study,a simple,rapid,sensitive,specific,accurate and stable POCT technique was developed by integrating LAMP reaction and microfluidic chip,rapid field detection of monkeypox and other infections in Africa provides technical support.Methods:This study developed a microfluidic LAMP method for simultaneous detection of EBOV,MARV,RVFV,LASV,YFV,VARV,C-MPXV,and W-MPXV based on a ten-channel microfluidic chip and a portable fluorescence detection system.The sensitivity and specificity of pathogens on the microfluidic chip were detected by using the best primer combination screened in Ep tube.At the same time,whether there is cross contamination on the adjacent channels on the microfluidic chip is verified.Finally,the microfluidic LAMP technique was evaluated using serum and skin swabs with pseudoviruses or standard plasmids as mock clinical samples and compared with gold-standard PCR results.Results:The results showed that,based on ten-channel microfluidic chip and portable fluorescence detection instrument,eight kinds of pathogens could be amplified within 65℃and 60 minutes.The results showed that the detection limit of(LOD)LAMP was 10~2-10~4copies/m L,which was 1-2 orders of magnitude lower than that of conventional PCR.Conclusion:The microfluidic LAMP technology developed in this study can specifically detect eight pathogens in 60 minutes.At the same time,the technology has the advantages of low cost,less reagent consumption.In the detection process,only 3μL template nucleic acid is needed for each reaction hole on the chip to carry out amplification reaction.In addition,a polymer film is used on the chip surface,while air intakes and vents are sealed with a jammed case to isolate them from the outside world,reducing the possibility of aerosol contamination.the technology has the advantages of low cost,low reagent consumption,low possibility of aerosol pollution,and the characteristics of miniaturization and integration,more suitable for resource-poor remote areas such as Africa,rapid multi-virus field diagnosis,timely treatment of emerging pathogens and prevent further spread of great significance.