Study On Rapid Quantitative Detection Technology Of Respiratory Viruses Based On Digital Isothermal Amplification Chip

Infectious diseases caused by respiratory viruses have seriously threatened human health.Positive or negative determination of infectious diseases is the top priority for early diagnosis,but accurate determination of viral load is also essential for determining the degree of infection and optimal course of treatment,monitoring the effectiveness of treatment,and distinguishing between active infection and carrier.At present,it is of great significance to study the quantitative detection technology of respiratory virus with stable performance,sensitive and rapid,which integrates sample processing and digital nucleic acid amplification.It can not only improve the detection efficiency and reduce the control difficulty,but also further improve the stability,accuracy and sensitivity of quantitative analysis.In this study,the Severe Acute Respiratory Syndrome Corona Virus 2(SARS-CoV-2)was used as the model.We have carried out research on rapid extraction of nucleic acid and rapid isothermal amplification for respiratory viruses,and developed a digital isothermal amplification chip which integrated viral nucleic acid extraction,reagent storage,digital division and amplification.In addition,a digital quantitative detection system integrating automatic gas-liquid control,constant temperature heating,electric displacement and fluorescence imaging acquisition was designed and manufactured.Rapid quantitative detection model was established based on respiratory tract samples,and the quantitative analysis of respiratory tract viruses from sample to result was realized.The main contents and results are presented as follows:1.Based on the principle of immiscible filtration assisted by surface tension,(IFAST),the IFAST system consisting of guanidinium hydrochloride(Gu HCl),Novec HFE 7500(HFE 7500)and eluent(come from commercial kits)was established for rapid extraction of viral nucleic acid from nasal swabs eluate.The lysis efficiency of Gu HCl against virus was evaluated,and the lysis efficiency exceeded 92.29%.Based on the finite element analysis,the factors affecting the interface stability of the IFAST system were explored,and the IFAST extraction chip was designed,optimized and fabricated according to the simulation results.The simulation results show that the interfacial tension and interfacial height affect the interface stability of IFAST system by affecting the critical pressure.The interfacial height is inversely proportional to the critical pressure,while the interfacial tension is linearly and positively correlated with the critical pressure.According to the simulation results,the interface scale of IFAST extraction chip was optimized.The height of the immiscible channel was set to 150μm so that the magnetic beads could be smoothly transferred during nucleic acid extraction and the immiscible interface could remain relatively stable.The effects of particle size,adsorption efficiency,magnetic responsiveness and dispersion of seven commercial magnetic beads on the extraction efficiency of IFAST were analyzed comprehensively.The recovery efficiency of four typical particle sizes of magnetic beads in the IFAST extraction chip was tested,and a mixed magnetic bead was selected for subsequent IFAST extraction efficiency analysis.The recovery efficiency of the mixed magnetic beads in the IFAST chip was(92.05±1.98)%,which was higher than the reported recovery efficiency of(84.9±2)%.The extraction efficiency of nucleic acid for graded diluted SARS-CoV-2pseudoviruses in IFAST chip were tested.The extraction efficiency was 88%-91%(from TE buffer)and 78%-85%(from nasal swab eluate),respectively,which was higher than the efficiency of commercial kits of 45%-67%(from nasal swab eluate).2.The genome structure and nucleotide sequence of respiratory virus SARS-CoV-2 were compared and analyzed.Finally,N gene was selected as the target gene of reverse transcription loop mediated isothermal amplification(RT-LAMP).Based on specific primer design and screening,reaction condition optimization,sensitivity and specificity analysis,a rapid and sensitive RT-LAMP amplification system for SARS-CoV-2 was established,which could quickly identify SARS-CoV-2 and its five variants of concern(VOC)within 30 minutes.The detection sensitivity of SARS-CoV-2(Wuhan-Hu-1)was 3.6×10~2copies/m L.The thermostatic drying analysis of the SARS-CoV-2 RT-LAMP system was performed based on the commercially available RT-LAMP premix Mix.It was found that there was no significant difference in sensitivity detection before and after drying of the reaction system.3.Based on the nucleic acid quantization process of respiratory tract samples,the structure design,optimization and preparation of integrated digital isothermal amplification chip were carried out.Sample nucleic acid extraction,digital division of the reaction system and amplification were all integrated in one chip.The integrated chip mainly included two functional regions:IFAST extraction region and digital loop-mediated isothermal amplification(d LAMP)region.A pneumatic valve structure was designed inside the chip to connect IFAST extraction and d LAMP amplification.By controlling the opening and closing of the pneumatic valve,the fluid in the chip could be self-introduced and self-digitized.According to the structure characteristics of the integrated chip,a quantitative detection scheme for respiratory tract samples was established.And rapid nucleic acid extraction,self-digital segmentation and absolute quantitative analysis of nucleic acid could be performed for large volume nasal swab eluate.Simulation analysis model were established to comprehensively analyze the performance,control parameters and fluid stability of the integrated chip.According to the simulation results,the internal structure parameters of the chip were optimized.The simulation results show that the interface of IFAST system is still stable when eluent is introduced into the microarray region by negative pressure.In order to reduce the difficulty of the preparation of the integrated chip,the flow path on the right side of the elution chamber was optimized to be 30μm in height and 350μm in width.In addition,through the simulation calculation,when the gas channel flation pressure is 500mbar,the pneumatic valve can effectively close the upper fluid channel.Through the fluid control test of lemon yellow solution,the accuracy of the simulation analysis results was verified,and the controllability,stability and rationality of the integrated chip were further verified.Based on the integrated chip,the digital segmentation rate and uniformity test,thermal stability test and biocompatibility test were carried out.When degassing the integrated chip with negative pressure for more than 10min,the sample digital segmentation rate could reach more than 99%.Using HFE 7500 with 1%d SURF as the segmentation oil could effectively improve the stability of the aqueous phase in the microchambers during heating and amplification.The digital amplification of SARS-CoV-2 RT-LAMP amplification system could be performed in the integrated chip.The contrast between negative and positive reaction signals were obvious after amplification,which indicates that the integrated chip,amplification system and oil segmentation had good biocompatibility.In addition,a data analysis model was established based on MATLAB software to statistically analyze the amplification results of negative and positive samples.4.In order to improve the detection efficiency,a digital quantitative detection system was designed and developed,which was used for the automatic control of gas and liquid,constant temperature heating,electric displacement and fluorescence imaging of the chip.Through the demonstration of system function requirements,component selection,module trial production and structure optimization,the digital quantitative detection system included a gas-liquid control module,a temperature control module,an electric displacement module and a fluorescence imaging module.The structure of the system was reasonable,compact and the volume was relatively small.Through functional verification,the system could reliably realize the stability control,digital isothermal amplification and fluorescence signal acquisition of the integrated chip.5.Based on the rapid and accurate quantitative analysis of SARS-CoV-2 in respiratory tract samples,the pre-embedding technique of SARS-CoV-2 RT-LAMP amplification system was studied.The amplification system was introduced into the chip in advance and dried at constant temperature,which could reduce the operation steps and control difficulty in the detection process and further improve the detection efficiency.Quantitative analysis of SARS-CoV-2 recombinant plasmid samples with gradient dilution based on the integrated chip were performed.The dynamic detection range was10~6copies/m L to 10~1copies/m L,spanning 6 orders of magnitude,which was better than the quantitative analysis results of typical commercial digital quantitative detection systems(Bio-RAD,QX 200).Moreover,the quantitative detection results by the integrated chip had a high linear correlation with the theoretical value(R~2=0.9986),and a high consistency with QX 200 in a certain range(R~2=0.9992).The rapid quantitative detection model of SARS-CoV-2 was established,including swab collection and elution,sample loading,nucleic acid extraction and quantitative analysis,which could realize the accurate quantitative detection of"sample in to answer out".The quantitative analysis of the artificial pollution samples showed good linear relationship between the detected value and the theoretical value(R~2=0.9958).The dynamic range of the detection was 10~5copies/m L to 10~0copies/m L,and the dynamic range was over 6 orders of magnitude.The lower limit of the detection sensitivity was6copies/m L.The detection time was about 45min,which was less than the detection time of QX 200(106min).