Establishment And Implement Of Novel Pathogen Detection Strategies

Pathogenic microorganism,also called pathogen,is defined as any microorganism that can cause disease to its host.The spread of pathogens is fast and wide,which seriously threatens human health and safety.Fast and accurate detection of pathogens can effectively prevent the spread of infectious disease and ensure the safety of public health.Traditional pathogen detection methods such as plate culture,PCR amplification have been widely used in clinical diagnosis,but they still have some limitations such as low sensitivity,high cost,and low portability,which unsuitable during the detection of low-concentration samples,mixed infections or unexplained infections.Since the 20th century,the acceleration of globalization and virus mutation have brought new challenges to the prevention of infectious diseases.Therefore,it is of great significance to develop new pathogen detection methods with the characteristics of high sensitivity,rapidity,good specificity,and simple operation.In this thesis,we take the nucleic acids and proteins of pathogens as the main research object.On one hand,combined with microfluidics,isothermal amplification,mass spectrometry,and CRISPR/Cas12a system,we have developed a series of new methods for the detection of pathogens such as HPV and E.coli O157:H7 based on their proteins or nucleic acids biomarkers.On the other hand,we have found potential protein biomarkers for the detection of pathogens such as A.flavus based on quantitative proteomics analysis.These methods provide new strategies for the efficient and rapid detection of pathogens.The main achievements are as follows:1.To improve the sensitivity and specificity of current nucleic acids detection methods,we have developed a new nucleic acid detection platform based on the CRISPR/Cas12a system.Previous studies have shown that Cas12a could tran-cleave ssDNA and has been widely used in nucleic acid detection.In this study,we have discovered and confirmed that Cas12a could tran-cleave DNA G-quadruplex(G4)and G-Triplex(G3).We have established the G-CRISPR platform based on this new discovery.Compared with the platforms using ssDNA as the reporter,our G-CRISPR method has obvious advantages in sensitivity.The detection sensitivity of unamplified and amplified plasmids can reach 50 pM and 0.1 aM,which are increased by 20 times and 10 times,respectively.These G4 and G3 with advanced space structure could significantly improve the sensitivity when used as new reporter in nucleic acid detection for the reason that they can bring more significant fluorescence changes before and after being cut.Through the detection of HPV(Human Papilloma Virus)in 27 clinical patient samples,G-CRISPR showed high specificity and accuracy,with a positive prediction and a negative prediction of 100%and 97.5%,respectively.The G-CRISPR nucleic acid detection platform is expected to be widely used for rapid detection of pathogens.2.To simplify the cumbersome operation of existing protein detection methods,we have introduced an naked-eye based quantitative platform for E.coli O157:H7 detection:EA-Sensor.For the first time,we demonstrate the combination of aptamerrecognition,hybridization chain reaction(HCR)-amplification and microfluidic chips for pathogen detection.We realized naked-eye based quantitative detection through converting the invisible E.coli O157:H7 concentration information into the indicator length information which is visible.EA-Sensor has a limit-of-detection(LOD)of 250 CFU/mL and has good performance in complex systems such as milk.EA-Sensor shows advantages of high specificity,easy operation,efficient amplification and visualized readout,which offers a favorable point-of-care tool for E.coli O157:H7 or other pathogen detection in resource-constrained settings.3.In view of the lack of suitable protein biomarkers for some pathogens,five Aspergillus flavus strains with different toxin-producing ability have been studied through quantitative proteomics based on high-resolution mass spectrometry.We have quantified a total number of 4,363 proteins,among which 1,045 proteins were differentially expressed between the high-and low-aflatoxin-yield A.flavus strains.These differentially expressed proteins mainly regulate toxin production through carbon metabolism pathways.According to the protein expression differences and protein location information,we have successfully screened potential protein markers such as RodA,RodB for rapid identification of high-and low-aflatoxin A.flavus strains.The molecular mechanisms underlying aflatoxin production has also been discussed.The quantitative proteomics analysis method provides a new strategy for the study of protein biomarkers in A.flavus,and can also be widely used in other pathogens.In summary,we have developed a series of new detection methods that can be used for pathogens detection,such as HPV,E.coli O157:H7 and A.flavus.These strategies cam be expanded to the detection of other pathogens,and we also expect these techniques could facilitate relevant diagnosis in clinic.