| In recent years,due to environmental changes,natural disasters,species diversity,large-scale use of antibiotics and other factors,infectious diseases caused by various pathogens continue to outbreak,such as African swine fever virus,Ebola virus,avian influenza virus and so on.In clinical medicine,pathogen infection is an important inducement of many diseases,such as hepatitis,tuberculosis,cancer and so on.Traditional pathogen detection methods are mostly only suitable for laboratory environment.They have the shortcomings of single detection target,complex operation,high requirements for environment and personnel,and are difficult to meet the needs of rapid pathogen detection.In this paper,a new method of multiplex nested solid-phase polymerase chain reaction(SP-PCR)amplification is proposed and established to solve the problem of rapid parallel detection of multiple pathogens.By constructing solid-phase PCR-Array chip and introducing nested PCR amplification method,the detection flux and specificity of multiplex detection are improved.Based on the technology of microfluidic chip,the rapid enrichment of pathogen nucleic acid on the chip is studied according to the different detection objects.With the method,DNA and RNA were extracted simultaneously on a chip,multiplex nested solid-phase PCR amplification and product detection were carried out to realize the integration,rapid and parallel detection of pathogens.Specific research contents include:(1)Research on Micro fluidic Solid Phase PCR Chip and Establishment of Reaction SystemIn order to achieve parallel detection of multiple pathogens in a single PCR reaction system without interference,we combined microfluidic chip with solid-phase PCR technology.We researched the effects of different solid-phase primer concentration and ultraviolet cross-linking time on the efficiency of solid-phase PCR,and produced solid-phase PCR-Array chip to improve the detection flux.Meanwhile,a new method of multi-nested solid-phase PCR amplification on microfluidic chip was proposed and established by introducing nested PCR amplification method.The effects of different free upstream and downstream primer ratios on the efficiency of nested solid-phase PCR were studied.The specificity and sensitivity of multi-nested solid-phase PCR-Array chip for simultaneous detection of multiple pathogen genes were tested using four pathogen plasmids.The results showed that under the optimized experimental conditions,the detection limit of multi-nested solid-phase PCR-Array chip was 10 copies/?L.The establishment of this method lays a foundation for the integrated rapid detection of multiple pathogens.(2)Design and experimental study of integrated microfluidic nucleic acid analysis chipIn this part,based on the established multiple nested solid phase PCR-Array chip,and taking advantage of the integration of microfluidic chips,a microfluidic nucleic acid analysis chip was proposed and designed based on the principle of magnetic bead nucleic acid extraction.The chip extracts bacterial DNA and virus RNA simultaneously,and solid PCR reaction.Four representative strong pathogens including Ebola virus,Crimea-Congo hemorrhagic fever virus(CCHFV),Bacillus anthracis and Brucella were selected as the research objects.Four positive quality control products containing pathogen specific gene sequences were constructed,and the quality control products were accurately quantified.The quality,sensitivity and repeatability of nucleic acid extraction were evaluated.The results showed that the sensitivity of bacterial DNA extraction by integrated microfluidic nucleic acid analysis chip was 102 CFU/mL,the sensitivity of viral RNA extraction was 104 copies/mL,and the relative standard deviation(RSD)of the sub-repeatability experiment was less than 2%.The results show that the integrated microfluidic nucleic acid analysis chip can achieve rapid and parallel detection of four pathogens with well specificity,sensitivity and repeatability.(3)Design and experimental study of integrated microfluidic HPV genotyping chipOn the basis of previous studies,we designs an integrated microfluidic chip based on the principle of nucleic acid extraction by heating pyrolysis,and integrates DNA extraction,nested solid-phase PCR amplification and product detection onto a chip.The design of pneumatic micro-valves and micro-pumps on the chip was improved,which had the functions of reagent mixing and storage,greatly reduced the complexity of the chip and reduced the size of the chip.Five high-risk HPV genotypes HPV16/HPV18/HPV31/HPV33/HPV58 positive quality control samples were used as test samples to evaluate the performance of integrated chip in extracting HPV DNA and the specificity,sensitivity and accuracy of detecting each genotype.The experimental results show that the chip can extract samples with103 copies/mL of HPV virus,and extract DNA concentration and initial HPV positive quality control products.The R2 value was 0.983,which indicated that the chip had good repeatability in extracting HPV DNA.20 clinical female cervical swab samples of HPV genotyping identification experiments were carried out on the integrated chip.At the same time,the traditional detection methods were compared with the international advanced instruments and equipment.The results showed that the detection results of integrated microfluidic solid-phase polymerase chain reaction chip were consistent with those of traditional methods,and the accuracy was 100%.The integrated chip greatly reduces the cost of reagents and detection time,and can complete the screening of five genotypes in one hour.In summary,aiming at different pathogen detection requirements,different types of integrated microfluidic nucleic acid analysis chips are designed.The chips integrate nucleic acid extraction,amplification and detection,which can realize high sensitivity,high specificity,fast and parallel detection of multiple pathogens.This research provided a high throughput and functional integration for the transmission,diagnosis and control of pathogenic infectious diseases. |
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