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Study on the PCR Detection Method and Health Risk Assessment of Enteric Pathogens in Water Environment

Author: ZhangChongZuo
Tutor: WangXiaoChang
School: Xi'an University of Architecture and Technology
Course: Environmental Engineering
Keywords: water environment enteroviruses enteric pathogenic bacteria PCR health risk assessment
CLC: X824
Type: PhD thesis
Year: 2008
Downloads: 559
Quote: 6
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Pathogenic pollution of water environment is one of the most serious problemsall over the world.Study on pathogenic pollution of water environment and healthrisk assessment is of great significance.Many uncertainty factors in health risk ofpathogens using traditional pathogenic microorganism indicator,polymerase chainreaction (PCR)with advantages of high sensitivity and specificity has showedsuperiority in detection of pathogens in medical science.However,the research aboutusing PCR to detection pathogens in water environment is limited at present.There islack of effective PCR detection method,and the study of health risk assessment on thebasis of PCR result is blank.Therefore,funded by the National Natural ScienceFoundation of China project "Study on detection of enteric viruses and pathogenicrisk assessment",this study was carried out to establish effective PCR detectionmethod of pathogens,to investigate the distribution and variation of pathogens presentin urban surface water and to characterize the human health risk associated withpathogens based on the PCR results.According to the characteristics of waterborne diseases and pathogenic pollutionin water environment in our country,enteroviruses were selected as typical entericviruses;Salmonella typhi,Shigella,Escherichia coli and Vibrio cholerae wereselected as typical pathogenic bacteria in water environment in this study.Thequalitative PCR and real-time fluorescent quantitative PCR detection method of thetypical pathogens were established.Using microporous filter adsorption-elution method to concentrate enterovirusesin water samples,the effects of many factors including nominal pore size,material offilter,the type of eluant and elution,and PEG concentration on virus recovery wereinvestigated and the concentration method was optimized.The results showed that cellulose mixed-ester microporous filter with nominal pore size of 0.22μm was superior to cellulose nitrate filter and nylon filter.Magnetism agitation was used for membrane elution and the optimal final mass concentration of PEG was 130g/L ineluant concentration step.Compared with talcum powder-diatomaceous earth layeradsorption-elution method,chemical flocculation precipitation method and talcumpowder-diatomaceous earth flocculation precipitation method,the modifiedmicroporous filter adsorption-elution method established in this study had more than70 percent of virus recovery in various virus inoculations(38~3800CCID50).Two pairs of universal primers of enteroviruses were designed by analyzing thehomology of the highly conserved 5’noncoding region nucleotide sequences of theenterovirus genome.Poliovirus type 1~3,Coxsackie virus type B3 were used asreference virus strains,RT-nested PCR method of enteroviruses was established.Compared with M-MLV transcriptase,AMV transcriptase was more suitable to thepractical application because both in surface water and sewage virus RNA all can betranscribed by AMV transcriptase.55℃annealing temperature and 2mmol/Lmagnesium ion was determined as optimized condition in first round PCR.ThisRT-nested PCR method had good specificity and high sensitivity (0.039CCID50),thusit was more suitable for detecting low concentrations of virus in environmental watersamples.Based on the RT-PCR of enteroviruses,real-time fluorescent quantitativePCR of enteroviruses was established.The recombinant plasmid was constructed asenterovirus DNA standard by cloning poliovirus cDNA into a pMD18-T vector.Optimized concentration of magnesium ion was as 4.0mmol/L,annealing temperatureas 55℃,and reading plate temperature as 85℃.The results showed that the detectionlimit of 4.62 genome equivalent copy (GEC)/μL,no cross-reaction of othermicroorganisms,wide dynamic linear relationship in the template range of 4.62GECto 4.62×109GEC (R2=0.997),and high precision of the intra-and inter-assayvariations lower than 2% and 5%,respectively.To the detection of typical pathogenic bacteria,qualitative PCR method andreal-time fluorescent quantitative PCR of Salmonella typhi,Shigella,Escherichia coliand Vibrio cholerae were established,respectively.Using standard strains ofSalmonella typhi,Shigella and Escherichia coli,the standard curve of real-timequantitative PCR,the dynamic linear relationship in the template range of 0.366CFU~3.66×105CFU,0.44CFU~4.4×105CFU,and4.56CFU~4.56×105CFU,respectively.Applying these PCR detection methods to surface waters (source of drinking water-Heihe River,landscape and recreational water-Xingqinghu Lake and BeihuLake,urban river-Chanhe River)and reference water-secondary effluent in Xi’ancity,the distribution and variation of typical pathogens in various environmentalwaters were researched by one year surveillance.It was found that enterovirusespresented in surface waters and secondary effluent,and the concentration obeyedlog-normal distribution.The average concentration of enteroviruses in Heihe River,Xingqinghu Lake,Beihu Lake,Chanhe River and secondary effluent was of 47GEC/L,127GEC/L,120GEC/L,422GEC/L and 746GEC/L,respectively.For HeiheRiver,most of enteroviruses positive samples and high concentration samplespresented in spring and summer.For Xingqinghu Lake and Beihu Lake,the period ofhigh concentration and high positive rate were in late of summer and early of autumn.Obvious fluctuation of enteroviruses concentration with change of season wasobserved in Chanhe River.For secondary effluent,early of September tomid-December and late of April to the end of May were two periods of highconcentration of enteroviruses.The fact that high similarity of nucleotide sequencesfrom various water samples indicated that the single species of enterovirusespresented in water environment.Salmonella typhi,Shigella,Escherichia coli,and Vibrio cholerae were detected by PCR method,and total bacteria,total coliform and fecal coliform were alsodetected by traditional culture method.The results showed that Vibrio cholerae wasnegative in all waters and the concentrations of other pathogenic bacteria were subjectto the lognormal distribution.The detection results of Salmonella typhi in samplesfrom Heihe River,Xingqinghu Lake and Beihu Lake were all negative.It was foundthat high concentration of Salmonella typhi and no obvious regularity of concentrationvariation in Chanhe River and secondary effluent.Shigellae were found in all waters.The concentration of Shigella was low before the summer,but it increased rapidly insummer.The concentration of Escherichia coli gradually rose from winter to summer.By analyzed the relationship between enteroviruses,enteric pathogenic bacteria,bacteriological index and physical-chemical index,the results indicated thatbacteriological index can not replace the detection of enteroviruses because of noevident correlation between them.In many waters,total bacteria count,total coliformand fecal coliform showed positive correlated with water temperature,turbidity andCOD,and negative correlated with DO.When the concentration of total coliform orfecal coliform was more than 103CFU/100mL,Salmonella typhi increased rapidly. Shigella was significantly positive correlation with bacteriological index,and theconcentration gradually increased with concentration of coliforms or fecal coliforms,but Shigella presented in the waters with low concentration of coliform.Based on the PCR results of enteric pathogens,Human health risk associatedwith pathogens in various waters was evaluated combined with exposure assessmentand dose-response analysis."Enteric pathogens of maximum risk" in various waterswas determined and requirement of risk control was also proposed.The result showedthat enteroviruses were enteric pathogens of maximum risk in Heihe River.Enteroviruses must be effectively removed to ensure the safety of drinking water.When virus removal reached 6-log,the probability of enteroviruses infection riskbelow acceptable annual risk level (10-4/a)was more than 90%.For Xingqinghu Lakeand Beihu Lake,enteroviruses were still major health risk source.When virusremoval was 4-log,the safety was more than 90%.For Chanhe River,major healthrisk was multiple.When direct as landscape and recreational water,health risk wasmainly caused by Shigella and Escherichia coli.Because of different variation ofenteroviruses and enteric pathogenic bacteria,water safety associated withenteroviruses was low when the removal of virus and pathogenic bacteria were 4-log.

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