| Background: Cervical cancer is a malignant tumor that occurs in the cervix,vagina and cervix,and is one of the most common gynecological malignancies.Human papillomavirus(HPV)infection is closely related to cervical cancer and its precancerous lesions.It is the most common sexually transmitted virus infection worldwide.HPV has identified more than 120 subtypes,and different subtypes can cause different clinical manifestations.According to the closeness of different subtypes to cervical cancer,they can be divided into high-risk HPV(hr HPV)and low-risk HPV(lr HPV).).For the detection of HPV,the traditional clinical methods use serological methods,Pap smears,liquid-based cell thin-layer technology and other detection methods,which are complicated and subjective,the results lack accuracy,and the specificity and sensitivity are not ideal.Therefore,it brings great difficulties to the clinical diagnosis and treatment of HPV.With the continuous development of molecular diagnostic technology,molecular biotechnology using nucleic acid probe hybridization has become the mainstream method of HPV typing and detection.The application of microarray-based reverse hybridization technology in HPV typing detection has higher sensitivity and specificity than traditional methods,and can be used to detect multiple samples and multiple subtypes at the same time.In recent years,the application of microfluidic technology in the field of molecular diagnostics has developed rapidly.Integrating multiple device functions required for microarray experiments on a palm-sized chip,the combination of microfluidics and microarray technology can achieve less sample and reagent usage and reduce the time required for incubation,and the high surface area to volume ratio in the nano-solution volume microchannel greatly improves the detection sensitivity.Objective: This study proposed a nucleic acid detection system based on a microfluidic chip,which is applied to detect 24 HPV genotypes in samples.In addition,we also optimize its detection process and conduct experimental evaluations on the detection performance of the optimized microfluidic system.Method:(1)Microfluidic chip design: Polystyrene is used as the main material to make the main body of the microfluidic chip,laminated with PCR tube for multiple PCR amplification,and finally,a microarray containing 24 HPV genotype detection probes was inserted for reverse dot blot detection.(2)Amplification primers were designed based on the conservative sequences in the L1 region of the 24 HPV gene sequences,and the upstream and downstream primers of each genotype were degenerate to determine 8 upstream primers and 7 downstream primers.At the same time,design detection probes to make DNA microarrays.(3)From May 2019 to January 2021,cervical swab samples were collected from 110 cervical disease outpatients in the Laboratory of the First Affiliated Hospital of the University of Science and Technology of China,and all samples were collected before the patients had undergone any treatment.(4)Screen the pump fluid pathways driven by PRD and PCRD,calculate the total amount of multiple pump fluids and compare the same pump fluid pathways on the same chip and different chip units,and calculate the overall pump fluid deviation of the micropump to evaluate this The pumping accuracy of the microfluidic system.(5)The nucleic acid extraction step is performed on the microfluidic chip with three different magnetic beads,and the nucleic acid is subjected to quantitative PCR detection,and the nucleic acid extraction efficiency is compared to determine the type of magnetic bead with the highest extraction efficiency.(6)Make microarrays with two differently modified HPV16 subtype probes of different concentrations,perform the hybridization process under different hybridization temperature and time conditions to optimize the hybridization conditions,and perform the hybridization process with different concentrations of HPV16 nucleic acid samplesto measure the hybridization sensitivity.(7)The 24 kinds of HPV subtypes national standard reference materials were serially diluted to different concentrations and mixed with HEK293 cells as simulated samples to test the detection limit of the method.(8)Use this method and a similar test kit approved by the National Medical Products Administration of China to test 110 clinical samples,and compare the test results of the two methods to evaluate the clinical performance of the method.Results: On the microfluidic chip constructed in this study,the nucleic acid of the sample was extracted by the principle of chemical cell lysis and silicon-based magnetic beads combined with nucleic acid,and then primers were added to start the amplification cycle.The amplified product was melted into single-stranded DNA and DNA at 95℃ The microarray was hybridized at 45°C for 10 minutes.Finally,the color developing solutions were added to perform color reaction,and the detection result is identified according to the color-developing result.(1)The selected PRD pump path is RR-SR,PCRD pump path is ER-MM1,the overall CV of PRD pump is 15.97%,and the overall CV of PCRD is 21.55%.(2)Nucleic acids extracted using three different brands of magnetic beads were compared by RT-PCR quantitative detection,and Magsibeads-Allround had the highest extraction efficiency in the microfluidic chip(65±5%).(3)Reverse dot hybridization at 45°C for 10 minutes is the optimal condition,and the hybridization sensitivity is 100 p M;the higher the concentration of probes used to make the microarray,the better the hybridization results,but the hybridization results of two different modified probes are not obvious difference.(4)Simulated samples containing 24 different concentrations of HPV genotype plasmids were used for detection,and the lowest detection limit of the microfluidic chip was calculated to be 103 copies/m L.(5)The comparison with the test results of the commercial kit shows that this method can be used for the detection of clinical samples in most cases.Conclusion: The microfluidic chip proposed in this study uses multiple PCR-reverse dot hybridization as the core principle to detect 24 HPV genotypes.It has high detection sensitivity for the detection of 24 HPV genotypes,and compared with the detection results of the same type of reagent products,it shows that the system has the potential to be applied to clinical sample typing detection. |
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