Applications Of High-performance Capillary Electrophoresis And Advanced Nucleic Acid Detection Technology In Sanitary Analysis

Part?Determination of acesulfame potassium in swimming pool by high performance capillary electrophoresisObjective:To establish a new method for the determination of acesulfame potassium(ACE),a new urine indicator in swimming pool,by solid phase extraction-high performance capillary electrophoresis,and to apply it successfully to the detection ACE in swimming pool water.Method:The fused silica capillary with uncoated inner wall(75?m i.d,60.2 cm of total length,and 50 cm effective length)was used.10 mmol/L sodium tetraborate(p H=9.30)was adopted as electrophoresis buffer at 24 k V separation voltage.The sample was injected for 20 seconds at detection wavelength being set at 226 nm.The swimming pool water samples were analyzed by capillary electrophoresi after filtered and enriched by solid-phase extraction.Results:The results showed that under the optimized conditions,there was a good linear relationship in the range of 0.20?100.00?g/m L,with the linear correlation coefficient of r=0.9998,and the detection limit was 50.0ng/m L.The relative standard deviation(RSD)was 1.8%(n=5).The spiked recovery was within the range 96.0%?103.6%.Conclusion:The method is sensitive,simple and reliable.It is suitable for the detection of acesulfame potassium in swimming pool water,and the result is satisfactory.Part?Detection of caffeine and its main metabolites for early diagnosis chromatographyObjective:To establish a new micellar electrokinetic capillary chromatography method for the determination of the levels of caffeine(CA)and its three main downstream metabolites–paraxanthine(PX),theobromine(TB)and theophylline(TP)in human plasma,and successfully apply it to the early auxiliary diagnosis of Parkinson's disease.Method:The fused silica capillary with uncoated inner wall(75?m i.d,60 cm of total length,and 50 cm effective length)was used.35 mmol/L phosphate–25 mmol/L SDS(p H=10.5)was adopted as electrophoresis buffer at 15 k V separation voltage.The sample was injected for 15 seconds at detection wavelength being set at 210 nm.The serum samples were directly analyzed after enriched by solid-phase extraction.Results:The results showed that under the optimized conditions,there was a good linear relationship in the range of 0.5?100.0?g/m L for CA,0.4?100.0?g/m L for TB,0.3?100.0?g/m L for PX and TP,with the linear correlation coefficient of r>0.9996,and the detection limits of the four targets were 7.5 ng/m L for CA,5.0 ng/m L for TB,4.0 ng/m L for PX and 4.5 ng/m L for TP,respectively.The recovery was within the range of 88.0%?105.9%.The relative standard deviation was 0.5%?8.1%(n=5).Conclusion:The method is sensitive,simple and reliable.It is suitable for the detection of caffeine and metabolites in plasma,and the result is satisfactory,suggesting that the present method could serve as a practical early diagnostic in PD patients.Part?Rapid detection of norovirus genogroup?in food,water and recombinase polymerase amplificationObjective:To establish a new method for rapid quantitative detection of norovirus genogroup?in clinical and environmental samples by real-time reverse transcription recombinase polymerase amplification.Method:Universal specific primers and Exo probe were designed and synthesized to hybridize to all genetic clusters of No Vs G?based on the conserved region at the ORF1–ORF2 junction of the genome,uses a portable isothermal amplification instrument Genie?to monitor the amplification results in real time,and the detection can be completed at 39°C for 20 min.Results:The RT-RPA method can specifically amplify the No Vs G?,but no amplification of No Vs G?virus,hepatitis A virus,hand-foot-mouth virus and adenovirus.Transcribed No Vs G?RNA was used as a template,and the detection limit was up to 1.66×10~2 copies/?L.Real samples analysis showed a sensitivity of 96%and specificity of 100%.Conclusion:This method is simple and rapid,and has strong clinical applicability,which provides a powerful support for the monitoring,prevention and control of norovirus and disease outbreak.Part?Simultaneous separation and quantification of hepatitis A virus,?using triplex droplet digital PCRObjective:To establish a triplex droplet digital PCR method for the simultaneous quantification of norovirus genogroup?,?and hepatitis A virus.Method:Referring to the standard ISO 15216-2(2019),the specific primers and probes of norovirus genogroup?(No Vs G?),genogroup?(No Vs G?)and hepatitis A virus(HAV)were synthesized.The ratio based probe-mixing multiplexing strategy was used,the constructed plasmid standards were applied to the method development,the concentration of the probes,the annealing temperature and the annealing/extension time of the PCR amplification program were optimized,and the dynamic range,specificity,precision and sensitivity of the method were also regarded as main parameters to evaluate the performance.Results:The optimal primer probe concentrations in the dd PCR reaction were 900 nmol/L and 450 nmol/L,the annealing temperature was 59°C and the annealing/extension time was 1 min.The multiplexed dd PCR showed a good linearity performance in the plasmid range between 4 to 500 copies/?L(20 to 2500 copies/20?L dd PCR reaction solution),with correlation coefficent r>0.99.The quantitative limit was 4 copies/?L(20 copies/reaction),the detection limit was:norovirus G?and hepatitis A virus 5 copies/reaction,norovirus G?7.5 copies/reaction.Conclusion:The results showed a good specificity with no amplification of other viruses,the multiplex dd PCR method established can be applied for simultaneous rapid quantification of norovirus and hepatitis in real samples.