| Background and ObjectiveThe COVID-19 pandemic once spread on a large scale around the world,and now it has entered a low-level epidemic stage.Rapid detection of the SARS-CoV-2 virus can help monitor the epidemic situation and normalize the early warning system.Isothermal nucleic acid amplification methods have become a promising technique in molecular diagnostics due to their faster reaction times,less equipment,and fewer manipulation steps than PCR.This study developed a centrifugal microfluidics-based multiplex reverse transcription recombinase polymerase amplification(RT-RPA)assay for endpoint fluorescence detection of the E,N,and ORF1ab genes of SARS-CoV-2.Methods1.Hydrophilic modification and surface characterization of centrifugal microfluidic chip surfaces:poly(methyl methacrylate)(PMMA)was modified with a 2%hydroxypropyl methylcellulose(HPMC)solution,and then the surface modification results were characterized by a series of measurements,such as contact angle measurement,surface free energy measurement,surface element ratio measurement,surface roughness measurement,and so on.2.Optimization and verification of the capillary valve’s burst frequency formula on the centrifugal microfluidic chip:based on the chip structure characteristics designed in this study,the burst frequency of the capillary valve was modified and optimized on the basis of the classical formula for the capillary valve’s burst frequency.The burst frequencies of six groups of parallel capillary valves with different parameters were calculated using two classical formulas and the optimization formula derived in this paper.Then,the burst frequencies of these capillary valves were measured,the correlation between the calculation results and the measurement results was analyzed,and the optimal solution was selected to guide the design of the capillary valve.3.Construction and performance evaluation of a micro-real-time fluorescent RT-RPA system:a 10μL real-time fluorescent RT-RPA reaction system was constructed for nucleic acid detection of the E,N,and ORF1ab genes of SARS-CoV-2 and an internal reference gene(the human ACTB gene).Commercial pseudoviruses and enterprise reference materials(inactivated viruses)were used to evaluate the sensitivity and specificity of the system.4.The design of a microscope slide-shaped microfluidic chip and the construction and performance evaluation of the slide chip RT-RPA reaction system:a microscope slide-shaped centrifugal microfluidic chip was designed and fabricated,and the reagent dry powder was embedded.Through the steps of sample addition,centrifugation,incubation,and result detection,the quadruple RT-RPA detection of the E,N,and ORF1ab genes of SARS-CoV-2and the human internal reference gene(ACTB gene)was realized.Commercial pseudoviruses and enterprise reference materials(inactivated viruses)were used to evaluate the sensitivity,specificity,repeatability,and reproducibility of the system.5.Clinical sample verification:nasopharyngeal swab samples from 33 clinical patients(including 20 COVID-19 positive samples and 13 COVID-19 negative samples)were tested by slide chip RT-RPA.The RT-PCR results were taken as the standard method to calculate the sensitivity(positive coincidence rate)and specificity(negative coincidence rate)of the slide chip RT-RPA method.At the same time,a Kappa consistency test was carried out to determine the 95%confidence interval of the above two coincidence rates and Kappa values.6.Stability of RT-RPA reagent pre-stored on the chip:Chips were stored at the recommended storage temperature of-20℃,room temperature of 25℃,and high temperature of 45℃ for 12 weeks,and the stability of the RT-RPA reagent was observed.Results1.The hydrophilic modification method of the centrifugal microfluidic chip surface was determined and optimized:the hydrophilicity of the chip surface was increased by spin coating HPMC film(the contact angle decreased from 72.3±0.8°to 37.3±0.9°),and the hydrophilicity remained stable for at least 6 months.After modification,the total surface free energy increased from 38.20±0.22 m N/m to 48.45±5.30 m N/m,the surface roughness(Rq)increased from 1.6±0.1 nm to 13.3±2.7 nm,and the ratio of surface oxygen elements increased from 10.18±1.81%to 22.07±0.03%.2.The burst frequency formula for capillary valves was optimized and verified:the correlation between the calculation results of two classical formulas and the optimization formula derived in this paper and the actual measurement results were analyzed,and the linear regression equation was obtained.The correlation coefficients(R~2)of the equation were 0.969,0.957,and 0.959,respectively,which had good goodness of fit.The regression coefficients(slope)of the equation are 0.937,0.489,and 0.958,respectively,and the optimization formula derived in this paper is closest to 1,indicating that the calculated value is closest to the true size,so this formula is used to calculate the burst frequency in subsequent studies.3.A micro-real-time fluorescence RT-RPA system was constructed:the analytical sensitivity of the E gene in a 10μL real-time fluorescence RT-RPA reaction was 19.06 RNA copies/reaction(95%CI:15.75-59.83),and that of the N gene and ORF1ab gene was 17.45RNA copies/reaction(95%CI:14.04-59.83).Through the specificity analysis of six kinds of human infection coronavirus and eight kinds of respiratory tract clinical manifestation virus,only the ORF1ab gene showed non-specific amplification on the SARS virus.Multiple sequence alignment analysis showed that the 10μL RT-RPA reaction system of the three target genes was also highly specific for the detection of the SARS-CoV-2 variants of concern(VOC).4.The microscope slide-shaped microfluidic chip and the slide chip RT-RPA reaction system were constructed:the whole chip reaction process can be completed in 30 minutes.The sensitivity of the E gene,N gene,and ORF1ab gene were 40,20,and 10 copies per reaction,respectively.Through the specificity analysis of six kinds of human infection coronavirus and eight kinds of respiratory tract clinical manifestation virus,only the ORF1ab gene showed non-specific amplification on the SARS virus.In the chip repeatability test,the average fluorescence intensity CV values of the E gene,N gene,and ORF1ab gene were0.29%,0.71%,and 0.80%,respectively.In the chip reproducibility test,the average fluorescence intensity CV values of the E gene,N gene,and ORF1ab gene were 0.50%,1.06%,and 1.74%,respectively.5.Clinical samples were tested.Compared with the results of real-time fluorescence RT-PCR,the positive coincidence rate of the E gene was 90%(95%CI:69.90-97.21),and those of the N gene and ORF1ab gene were 95%(95%CI:76.39-99.11).The negative coincidence rate of the three genes was 100%(95%CI:77.19-100).The Kappa values of the N gene,E gene,and ORF1ab gene were 0.937,0.876,and 0.937,respectively,indicating that the results of the two methods were consistent.6.The stability test showed that the pre-stored RT-RPA reagents stored on the microfluidic chip for up to 12 weeks at-20℃ and 25℃ and for up to 4 weeks at 45℃ had no appreciable impact on assay performance.ConclusionThe centrifugal microfluidic chip for nucleic acid detection constructed in this study has the characteristics of being fast,accurate,on-site,and multiple,which would contribute to detecting COVID-19 patients in low-resource settings and point-of-care testing(POCT)and,in the future,could be used to detect emerging new variants of SARS-CoV-2.It also provided valuable experience for subsequent high-throughput,larger-area chip designs for various pathogens. |
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