Research On Rapid Detection Technologies For Foodborne Pathogens And SARS-CoV-2

Reseach on rapid detection technologies for foodborne pathogens and SARS-CoV-2Environmental pathogens have been threatening public health,the economy,and social stability.Foodborne pathogens,particularly Escherichia coli O157:H7,Staphylococcus aureus and Vibrio parahaemolyticus,have received the most attention because they threaten food safety and easily cause outbreaks.Accurate,rapid and reliable detection methods are urgently needed to reduce the hazards and contain the potential spread of foodborne pathogens.Currently,the common detection methods for these pathogens include conventional culture-based methods,molecular biology methods represented by PCR technology,and immunological methods represented by ELISA technology.However,these methods require tedious multi-step protocols,advanced instruments and well-trained personnel,which significantly hinder their application in resource-limited regions and during public health emergencies.Therefore,to improve on detection methods,I combined advantageous features of emerging technologies including immunological technology,nanotechnology,molecular biology,and microprocessing technology,and developed on-site rapid detection methods.During my Ph.D.program,coronavirus disease 2019(COVID-19)pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)ravaged the world.In the absence of effective vaccines and therapeutics,rapid and reliable detection of SARS-CoV-2 was critical to the mitigation and control of the pandemic.There was an urgent need for the development of accurate and efficient methods for detecting the virus.Therefore,based on my accumulated knowledge and experience in detecting foodborne pathogens,I developed a rapid method for the detection of SARS-CoV-2 and evaluated its performance.Section 1.Paper-based ELISA for the detection of Escherichia coli O157:H7In this section,the chitosan-glutaraldehyde modified filter paper was applied to replace the traditional ELISA plate,and the indirect ELISA method was performed on the filter paper.The detection results were interpreted according to the color change of the filter paper.The detection limit of this method for Escherichia coli O157:H7 was1×10~4 CFU/m L,which was better than the traditional ELISA method.And the specificity of this method was excellent.Also,the result of this method was able to be obtained through a smart phone without decreasing the detection sensitivity.Using this method to detect mock food samples,within the concentration range from 1×10~4CFU/m L to 1×10~7 CFU/m L,a linear regression curve was obtained:y=3.4145x+8.1657,and the correlation coefficient R~2 was 0.9828.Compared with the traditional ELISA method,this method has the advantages of low detection cost,short detection time,minimum instrument requirement,fewer reagents,and better sensitivity.The method is suitable for on-site detection applications.Section 2.Rapid detection method for Staphylococcus aureus based on dual-functionalized gold nanorod probe,magnetic enrichment and phenolphthalein test paperIn this section,a dual-functionalized gold nanorod probe modified with urease and yolk antibody was prepared by harnessing electrostatic adsorption,so that it was able to specifically recognize pathogens as well as possessed urease activity.In addition,a non-specific ferro ferric oxide magnetic bead was synthesized,which was able to effectively enrich various bacteria indiscriminately.The above two kinds of nanoparticles were incubated with Staphylococcus aureus,forming immunocomplex which was then magnetically separated.Finally,a urea solution was added to the supernatant.Ureases catalyzed the decomposition of urea,which resulted in an increase in the p H value.The increase in the p H value was detected by using a phenolphthalein test paper which underwent a color change from white to pink.The characterization results showed that the performance of the prepared dual-labeled gold nanorod probe was better than that of the conventional dual-labeled gold nanosphere probe.Also,under the observation of electron microscope,the non-specific magnetic beads efficiently and tightly combined with Staphylococcus aureus.The optimized detection method had a detection limit of 1×10~3 CFU/m L with good specificity.By testing contaminated cabbage and beef samples,the practicability and reliability of the method were verified.This method is convenient and time-saving without sacrificing sensitivity and specificity,and the entire detection process can be completed within 20 minutes.The detection result can be visually read without relying on any instruments.Thus,it is suitable for field test applications.Section 3.Rapid detection method for Vibrio parahaemolyticus based on MD-LAMP amplification and self-priming compartmentalization microfluidic chipIn this section,the SYBR Green dye and HNB dye were combined with the loop-mediated isothermal amplification,namely Mixed-Dye LAMP(MD-LAMP).The MD-LAMP amplification was applied on the self-priming compartmentalization microfluidic chip to realize quantitative detection of Vibrio parahaemolyticus.The self-priming compartmentalization microfluidic chip consisted of 1,056 micron-level reaction chambers and was made of PDMS material.Taking advantage of unique properties of the PDMS,the MD-LAMP reaction mixture was able to self-disperse into each micro-reaction chamber on the chip.After completing MD-LAMP reaction,by counting the number of green fluorescent spots,the quantity of Vibrio parahaemolyticus in the sample was quickly calculated.The experimental results showed that using MD-LAMP and microfluidic chip significantly improved the detection sensitivity,so that the detection limit of this method reached 1×10~3 CFU/m L.The method had good specificity.The test results of mock food samples indicated that the recovery rate of this method was between 93%and 104%,and the relative standard deviation was less than 9.3%,proving that the method was robust and reliable.The microfluidic chip used in this method is small and portable,which reduces transportation and storage costs.In addition,this method realizes autonomous sample injection avoiding the use of any syringe pump,which further reduces the operating cost.So,it is suitable for on-site detection applications.Section 4.Rapid detection method for multi-pathogens based on MD-LAMP amplification and self-priming PDMS/paper hybrid microfluidic chipIn this section,filter paper was combined with PDMS microfluidic chip,and a self-priming PDMS/paper hybrid microfluidic chip,termed SPH microfluidic chip,was developed.By using SPH chip,multiplex foodborne pathogens were able to be detected simultaneously.This chip achieved functional partitioning on a single chip by loading different nucleic acid primers on filter papers in different reaction chambers.Similar with last section,MD-LAMP technology was also applied on SPH chips to achieve accurate readout of test results.In this study,Staphylococcus aureus and Vibrio parahaemolyticus were selected as representative foodborne pathogens for detection.The experimental results showed that the detection limit of this method for plasmid templates containing the conservative nucleic acid sequence of Staphylococcus aureus was 2.15×10~1 copies/?L,and the detection limit for Vibrio parahaemolyticus plasmid templates was 2.09×10~1 copies/?L.Both of them were able to be tested individually or simultaneously on an SPH chip.This method possessed good specificity and no cross-contamination occurred among reaction chambers.The use of the SPH microfluidic chip not only reduces the work intensity of the operators,but more importantly,it doubles the detection efficiency and saves time for quickly responding to public health emergencies.Section 5.Single-tube rapid detection method for SARS-CoV-2 based on RT-LAMP amplification and CRISPR technologyIn this section,a single-tube assay for SARS-CoV-2 was described.This assay combined advantages of RT-LAMP for isothermal exponential amplification and CRISPR technology for specific detection.In principle,the target sequence of viral RNA was reverse transcribed and exponentially amplified by RT-LAMP.The specifically amplified target sequence was recognized by CRISPR g RNA and Cas12a.The activated Cas12a cleaved nucleic acid reporter molecules,producing a visible fluorescence signal.In practice,the temperature-incompatibility between the RT-LAMP reaction and CRISPR reaction was overcome by separately placing the RT-LAMP reaction mixture at the bottom of the test tube and the CRISPR reagents inside the cap of the tube.After 30 minutes of RT-LAMP reaction,simple inverting and wrist-flicking the tube mixed the Cas12a reagents with the amplicon generated by RT-LAMP.Experimental results showed that the detection limits of this method for the N gene and E gene of SARS-CoV-2 were 30 copies/?L and 45 copies/?L,respectively.Applications of this method to the detection of nucleic acid extracts from 100 clinical samples demonstrated the clinical specificity of 100%(detecting all 50 negatives),and the clinical sensitivity of 94%(detecting 47 positives of 50 total positive samples).This method has been developed into a portable kit consisting of reagents and simple apparatus.With assay instructions,and even in the absence of trained personnel,rapid SARS-CoV-2 screening can be completed within 50 minutes.