Research Of High-Throughput Nucleic Acid Solid-Phase Extraction Process Mechanism And The Applications In Pathogen Diagnostic PCR

Diagnostic PCR is an important nucleic acid（NA）based analysis tool in the fields of disease diagnosis and control,food safety,forensic medicine,and so on.Compared with the methods based on enzyme or antibody,it has a lot of advantages including high modification flexibility,high detection sensitivity,and can obtain the results faster and earlier.However,diagnostic PCR limited the use in modern laboratory mainly due to the cumbersome and time-consuming NA preparation extraction from the biological samples.Solid-phase extraction（SPE）has been accepted as a more promising approach for high efficiency NA separation because of the advantages of minimal hazardous chemicals,fewer and easier manual manipulations,automation capacity and enhanced throughput when compared to conventional liquid-phase extraction（LPE）.The mainstream high-throughput and automatable SPE is magnetic nanoparticles（MNPs）based method because of the NA-binding capacity and superparamagnetism characteristics of MNPs,which eliminates the need for centrifugation and electronic equipment.Although most of the MNP-based separations are fast（generally less than 15 min）and various available commercial kits have expanded rapidly,they still cannot meet the challenges of diagnostic PCR to use outside the laboratory for large-scale epidemiological investigation.NA extraction from biological samples include sample lysis and NA separation.Lysis of large-scale samples simultaneously now remains cross steam-contamination due to heating,long-time duration,complicated pretreatment and pipetting,PCR inhibitor residue and so on,which severely restricts the throughput and timeliness of the entire NA extraction process,and affects the accuracy of downstream diagnostic PCR.Besides,there have rare knowledge about the separation kinetic mechanism of MNP-based separation,such as,sedimentation and migration effects,performance of different surface modified MNPs with various size,and so on.While,the magnetic-free SPE still remains labor-intensive and low throughput,due to the cumbersome separation rely on multi-step centrifugation or pipetting,which is the key bottleneck.Herein,with high-throughput and automatable diagnostic PCR for practical routine use purpose,research on high-throughput NA solid-phase extraction process mechanism and applications in pathogen diagnostic PCR is introduced here.The contents and conclusions are as follows.（1）Study on the high-efficiency lysis process of biological samples.In order to promote the tedious and time-consuming lysis process,a low-cost,non-toxic alkaline solution was used to develop a room temperature lysis process performed on 96-well plate.Further combined with the MNP-based separation,a whole-process high-throughput NA extraction method called ALMS-extraction was established.Then,the method was applied for genomic DNA extraction from tussah tissue samples and dried blood spot samples,and its timeliness,extraction quality and anti-interference performance were evaluated.Results showed that,the alkali lysis is highly-efficient and fast（minimize to 5 min）.ALMS-extraction can isolate 96 samples simultaneously in minimize 20 min and has high NA extraction quality.It can overcome the key PCR inhibitory factors of the tissue samples and has high NA extraction quality.The entire extraction was under room temperature,there was low cross steam-contamination risk and had high PCR diagnosis accuracy and sensitivity.In conclusion,high-efficiency alkaline lysis process is proved to effectively improve the overall efficiency,whole-process throughput and NA extraction quality.This work provides a guidance for advanced optimizing and establishing template preparation in diagnostic PCR.（2）Research of the separation kinetic mechanism of MNP-based SPE.Aimed to enhance the selection of MNPs and illustrate the separation mechanism for effective extraction of NAs from complex biological samples,hydroxy-,amino-and carboxyl-functionalized MNPs（MNPs-OH,MNPs-NH₂ and MNPs-COOH）with four different particle sizes were fabricated to investigate the hybrid NA separation performance from animal tissue samples.Molecular dynamics（MD）simulation was introduced to predict the affinity action of MNPs with NA and typical impurities.Details of the kinetic separation behavior and NA separation performance were then illustrated.MD simulation results showed MNPs-NH₂ had a stronger affinity with NA,and in most experimental separation systems,NA separation yield via three kinds of MNPs showed a following order of MNPs-NH₂>MNPs-OH>MNPs-COOH.MNPs of different sizes had significantly different sedimentation and dispersion features,leading to diverse separation results.Combining with the proper lysis system,an optimized NA separation method at room temperature with high-throughput and low steam-contamination risk was developed as a promising approach for large-scale NA extraction simultaneously.This work could provide a theoretical guidance for the fabrication strategy of advanced MNPs and the enhancement of NA separation from complex biological samples.（3）Research of high-throughput magnetic-free SPE.In order to solve the bottleneck problem of lack fast separation driving force in magnetic-free SPE,with the precision manufacturing advantage of 3D printing technology,an integrated polyacrylic acid（PAA）comb-like separator with hybrid micro-structure was fabricated via 3D printing technology for high throughput,convenient and magnetic-free NA separation.Then,characterization analysis of structure,element and stability were carried out and NA extraction process,mechanism and performance were discussed in details.Results showed that,the separator has ideal printing accuracy and stability in the thread and groove structures,and can be reused with robust separation performance.The NA extraction yield enhanced by the hybrid micro-structures on the interface of the separator,benefiting by the increased specific surface area and enhanced interface affinity because of the enriched carboxyl groups on the surface.The NA extraction yield is comparable to that of commercial MNP-based extraction under optimized condition.The developed 3D-PAA method has advantages of very short separation time（minimized to 1 min）,no requirement of additional technical equipment（magnetic-free）and no intensive labors（no pipetting and centrifugation）.The NA amplification results illustrated that NA isolated via the separator could meet the requirements for further biological applications.It is promising in the field of high-throughput diagnostic PCR.（4）Applications of high-throughput SPE Technologies in pathogen diagnostic PCR.High-efficiency lysis process promoted MNP-based ALMS-extraction,and the newly established 3D-PAA method were further introduced for the practical application of PCR detection（ALMS-q PCR and 3D-PAA method for short）of microsporidium and plasmodium.Results showed that,ALMS-q PCR has significant higher sensitivity than conventional methods.In the pébrine control program,detection rate of ALMS-q PCR is higher than that of microscopy up to 10.8 times and the advantage is more significant in the groups with low infection level.It is possible to advance the testing period and find cross-infected microsporidium species in the wild field,so as to find the infections more completely and cut off transmission more effectively.While for plasmodium detection,ALMS-q PCR showed much higher infection detection rate than conventional nested PCR and eliminates the time-consuming gel electrophoresis in nested-PCR method.Besides,experiments confirmed that the 3D-PAA method can effectively realize the q PCR diagnosis of microsporidium and the nested PCR diagnosis of plasmodium.Based on its advantages of rapid,convenient and high-throughput,it is expected to be used for large-scale pathogen diagnostic PCR and epidemiological investigations.To sum up,this work has proved that enhanced highly-efficient lysis process of biological samples can efficiently promote the MNP-based NA extraction.The kinetic mechanism of MNP-based NA separation has preliminarily revealed.The promoted MNP-based NA extraction and the newly developed magnetic-free method were applied in diagnostic PCR and proved the feasibility,stability and advantages for high detection sensitivity.This study solves the problems in the template preparation of diagnostic PCR,and is of great significance to promote diagnostic PCR to be used out of the laboratory and realize the routine clinical application.There are broad application prospects of advanced diagnostic PCR in the fields of food safety,forensic detection,epidemic detection,disease control of agriculture and fishery,and so on.