Pathogen Rapid Detection Chip Research

Point-of-care testing(POCT)diagnosis technology is developing rapidly,especially in clinical diagnosis and other analytical fields.Its main features are the ability to quickly detect samples in real time,simple sample preparation,replacement of frequent pipetting with pre-filled reagents,user-friendly operation and instant results so that it can achieve "sample in,results out",especially in areas with poor medical environment and limited resources,showing great application potential.This paper designs and manufactures an integrated nucleic acid detection chip based on magnetic bead nucleic acid extraction and microfluidic technology,which can automatically extract nucleic acids including but not limited to 10 samples at the same time through the magnetic control system within 20 minutes.without further nucleic acid transfer,can be directly placed in the insitu amplification instrument for amplification procedures.Based on the principle of nucleic acid extraction by magnetic bead method,the chip stores the reagents required for detection(nucleic acid extraction reagent,cleaning reagent 1,cleaning reagent 2,PCR buffer)in multiple reaction chambers filled with mineral oil,through microfluidic.The cooperation of the control chip and the automatic displacement platform,the magneti c beads traverse each reaction chamber and microchannel to complete the mixing of reaction reagents and nucleic acid transfer in nucleic acid extraction,and realize the automatic extraction of nucleic acid in the sample.This operation mode that does not require fluid-driven greatly simplifies the current mainstream nucleic acid extraction methods,so that it can meet the needs of dozens of sample processing at one time,and the sample nucleic acid extraction and amplification are integrated on one chip to avoid the occurrence of crosscontamination caused by operation.The main work of this paper is as follows:1)A microfluidic chip that can be pre-filled with reagents required for detection is designed.The chip has 10 longitudinally arranged reaction unit arrays.At the same time,according to the detection requirements,a multi-target detection chip was designed and manufactured on the basis of the chip,which can divide the magnetic bead particles carrying nucleic acid into three parts as much as possible,and enter into three different detection systems respectively,which can be used for the three nucleic acid target sequences of the sample to be tested are simultaneously detected.2)An automated displacement platform for nucleic acid extraction is built,which can control the XY axis movement of the permanent magnet array.The magnet drives the magnetic beads to reciprocate continuously in the reagent to make the reagent reaction and mixing more fully,and realize the magnetic control method to complete the nucleic acid extraction automated.3)A series of optimizations and tests were made on the chip in terms of structure,surface properties and reagent selection.The experiments verified the performance of the chip in various aspects such as nucleic acid extraction efficiency,PCR amplification inhibitor removal ability and detection limit.In particular,a series of experiments were carried out to verify the ability of the chip to clear high concentrations of inhibitors in the sample.The aqueous dye experiments proved that even if high concentrations of dyes were added,the final concentration of dyes remaining in the PCR buffer was only<0.01%.The addition of amplification inhibitors(SDS,EDTA and Urea)also proved that the chip has a good inhibitor removal ability,thus ensuring the stability of the detection results in practical applications.4)Two respiratory pathogens,2019-n Co V and Mycoplasma pneumoniae,were detected using the chip.2019-n Co V detected its ORF-1ab gene.Two macrolide drug resistance mutation sites and conserved gene P1 were detected in Mycoplasma pneumoniae,A2063 G and A2065 G.Meanwhile,in order to verify its applicability to other sample types,H1975 cells were selected here and their EGFR exon 21 L858 mutation was detected.The results show that the integrated detection chip constructed in this paper can realize the lysis of biological samples,nucleic acid purification and on-chip amplification,with high detection sensitivity(4-5 copies/reaction),easy to operate,and is useful for realizing the detection of pathogens and other biological samples.Fast and highly sensitive detection has important reference significance.