大肠杆菌rep-PCR指纹图谱分析在粪便污染源示踪上的应用
摘要
微生物源示踪(microbial source tracking, MST)是90年代初美国学者提出一种利用环境中的微生物生化特性、遗传多样性或者其特异性代谢产物确定它的宿主来源,从而识别污染水体中粪便污染源的新的监测技术。主要用于应对人畜粪便对水域环境污染日益加重且久治无效的局面,解决将非点源粪便污染转化为可以控制的点源粪便污染。本课题采用MST检测技术中的rep-PCR技术和电泳技术通过对不同粪便污染源中指示因子(E. coli)的定性分析,总结指纹图谱规律,检验rep-PCR方法是否适合应用于我国水质监测,最终为国家管理部门识别、控制粪便污染源提供科学的理论基础。
目的
分析不同粪便来源的大肠杆菌rep-PCR指纹图谱规律,检验rep-PCR方法识别粪便污染源在我国水质监测中是否适用。
方法
本课题大肠杆菌样品采用前期大连海事大学从实验海域、陆源已知污染源分离、生化鉴定纯化后得到的629株大肠杆菌菌株,其中牛源128株,人源159株,鸡源111株,狗源120株,猪源111株；选用E.coli标准菌株ATCC25922为对从样品中分离出的E.coli进行阳性对照。
选择合适的E.coli基因组DNA提取方法。取等量样品,分别用酚／氯仿抽提法、CTAB法改良、CTAB法、液氮法、经典SDS方法、试剂盒法提取大肠杆菌基因组DNA,用紫外分光光度计(260nm,280nm)分别测定其OD值,进行定性、定量分析。确定合适的提取方法,保证有足够DNA模板进行PCR。
rep-PCR反应条件和电泳条件的摸索。对文献中查到的rep-PCR条件进行改良,改变PCR条件、模板加入量、引物浓度等,确定最佳反应条件。
ALPHAVIEW 2.0.0软件数据处理。采用ALPHAVIEW 2.0.0软件分析电泳结果,得到电泳结果图片中条带转化为以数字信息,以DNA marker为标准拟合出一条曲线,以此计算出待测菌株条带DNA片段长度,并以标准菌株的信息为对照验证每次结果的一致性。
指纹图谱分析。按牛、人、鸡、狗、猪不同来源分类,选取5000bp～100bp之间的条带进行分析,分别依照对数曲线确定每段的组距,共分128组,对电泳结果进行分段统计,扩增产物中分子量不同的DNA条带作为多态性状,分子量相同的条带作为一个相同性状,有性状记为1,无性状记为0,总结各来源大肠杆菌rep-PCR共有条带和特征性条带。
对rep-PCR结果进行统计分析。进行聚类分析,建立聚类分析树形图；并分别按照五种来源(牛、人、鸡、狗、猪),两种来源(人、动物),四种来源(牛、鸡、狗、猪)进行判别分析,检验rep-PCR快速鉴别的效果。
结果
考虑细菌基因组DNA提取结果和操作简便性,选择试剂盒法提取样品DNA,为进一步PCR提供足量可靠的模板。
rep-PCR反应体系总结：引物1R(10μM) 2μl、引物21(10μM) 2μl、2×SuperMix 25μl、DNA模板50 ng、加去离子水至50μl；PCR仪反应程序：50μl扩增体系,95℃预变性7 min,94℃变性30s,49.4℃退火1 min,72℃延伸8 min,重复30个循环,最后72℃再延伸16 min。
五种来源大肠杆菌在分子量581～590 bp、1251～1300 bp、3001～3200 bp处存在共有条带,不同种动物粪便中大肠杆菌各自具有特征性条带。
牛源大肠杆菌独有的特征性条带在分子量801～810 bp、1451～1500 bp、2201～2300 bp、2900～3000 bp处；人源大肠杆菌独有的特征性条带在分子量651～660 bp、771～780 bp、1451～1500 bp、2301～2400 bp处；鸡源大肠杆菌独有的特征性条带在分子量601～610bp、691～700bp、2301～2400bp处；狗源大肠杆菌独有的特征性条带在分子量441～450 bp、521～530 bp、771～780 bp、2901～3000 bp处；猪源大肠杆菌独有的特征性条带在分子量511～520 bp、651～660 bp、1451～1500 bp、2501～2600 bp处。
对样品扩增结果进行聚类分析(SPSS 16.0),得到树形图显示分层聚类关系；将样本按照牛、人、鸡、狗、猪不同来源分组,进行判别分析(SPSS 16.0),五组有显著差异(P<0.01),狗源、鸡源、人源、牛源、猪源的RCC值分别为96.7%、98.2%、93.6%、93.7%、92.5%,ARCC值为94.8%；
将四种动物宿主(狗源、鸡源、牛源、猪源)来源样品合并为动物组,人源样品归为一组,两组显示出显著的差异(χ2=440.246,P<0.01),人源RCC为86.7%,动物组RCC为90.1%,ARCC下降到89.2%。
分析四种动物源(狗源、鸡源、牛源、猪源)大肠杆菌图谱,四组有显著差异(P<0.01),狗源、鸡源、牛源、猪源的RCC值分别为96.7%、94.5%、96.9%、96.2%,ARCC值为96.1%。
结论
本课题结果显示：不同粪便来源大肠杆菌rep-PCR产物电泳结果存在共有条带,和各自的特征性条带,并且rep-PCR指纹图谱分析方法可以将在我国海域中采集样品中的大肠杆菌正确分组,能达到污染源示踪的要求,适用于我国水质监测。
For the demand to solve fecal pollution problem in water environment, Microbial Source Tracking (MST), as a new monitoring technology for identifying sources of fecal pollution in water, had emerged in 1990s in US. Using their biochemical characterristics, genetic diversity and its special metabolites to determine their host of the source, thereby identifying fecal pollution sources in water pollution. MST mainly used to deal with the situation that human or animal fecal pollution in the waters increasing and difficult to control, change the non-point pollution into point pollution which can be effective controlled. This subject adopts rep-PCR technology and electrophoresis technology, to qualitative analysis source indicator (E.coli) which collected from different pollution source. Summarizing the fingerprints to test rep-PCR applied to the water quality monitoring in China,in order to provide useful reference to the experts in this field and promote the research and application of MST in our country.
Objective
Summarizing the rep-PCR DNA finger prints analysis of E.coli, and detecting the applicability of rep-PCR method in the water quality monitoring in differentiating fecal pollution source.
Methods
A collection used for this study was collected from experiment maritime space and terrigenous area by researcher of DaLian Maritime University. The collection contained 629 fecal E.coli isolates from humans (159), cow (128), chickens (111), dogs (120) and pigs (111). The study used ATCC25922 as positive control.
To select the best method for extracting the genomic DNA of E.coli. Extracting the genomic DNA of E.coli from equal samples with 5 methods (Phenolic/chloroform, improved CTAB method, CTAB method, the liquid nitrogen, SDS method, and DNA extraction kit method). Using ultraviolet spectrophotometer to measuring OD of DNA templates for qualitative and quantitative analysis. Selecting appropriate methods and ensuring there are adequate DNA template for PCR reaction.
Selecting the best reaction conditions and electrophoresis conditions. Change the conditions of rep-PCR method from literatures, such as PCR conditions, template, concentration of primers for determining the best condition.
Using ALPHAVIEW 2.0.0 software to change the image informations to digital informations. Drawing curve with DNA marker as the standard, to calculate DNA fragment length and use the positive control to test results.
Fingerprint analysis. Dividing the samples into different classifications based on different source (human, cow, chicken, dog and pig). Choosing the bands between 5000bp-100bp for analysis. According to the curve, dividing into 128 groups to analysis the electrophoresis results. Summarizing the common bands and unique bands of rep-PCR fingerprints.
Statistical analysis of rep-PCR DNA fingerprints. The rep-PCR DNA fingerprints were subjected to hierarchical cluster analysis(SPSS 16.0). A dendrogram was constructed by using SPSS. Discriminant function analysis was also performed with the cluster analysis results to find out the number and percentage of isolates from each known source that were classified in each source category.
Results
Considering the genomic DNA results of 5 methods and operation convenience, selecting DNA extraction kit method as DNA extraction method to provide enough sample for PCR reaction.
Rep-PCR mixture:Primers 1R(10μM) 2μl, Primers 21(10μM) 2μl,2×Super Mix 25μl, DNA sample 50ng, up to 50μl with deionized water; Rep-PCR reaction process:95℃for 7 min, and this was followed by 30 cycles consisting of 94℃for 30s,49.4℃for 1 min,72℃for 8 min. The reaction was terminated with an extension step consisting of 72℃for 16 min.
Most isolates'rep-PCR fingerprints contain common bands in molecular weight of 581～590 bp,1251～1300 bp and 3001～3200 bp. Cow isolates'rep-PCR fingerprints contain unique bands in molecular weight of 801-810 bp,1451～1500 bp,2201～2300 bp and 2900～3000 bp; Human isolates'rep-PCR fingerprints contain unique bands in molecular weight of 651-660 bp,771～780 bp,1451～1500 bp and 2301～2400 bp; Chicken isolates'rep-PCR fingerprints contain unique bands in molecular weight of 601～610 bp,691～700 bp and 2301～2400 bp; Dog isolates'rep-PCR fingerprints contain unique bands in molecular weight of 441～450 bp,521～530 bp,771～780 bp and 2901-3000 bp; Pig isolates'rep-PCR fingerprints contain unique bands in molecular weight of 511-520 bp,651～660 bp,1451～1500 bp and 2501～2600 bp.
The cluster analysis was conducted with the rep-PCR DNA fingerprinting patterns of 621 fecal E.coli isolates after removal of the clonal isolates. The dendrogram can not produced obvious clusters. A discriminant function analysis was performed on the cluster analysis results to evaluate how accurately the rep-PCR DNA fingerprints were anle to predict a host source. Dog, chicken, human, cow and pig isolates were highly classified with a RCC of 96.7%,98.2%,93.6%,93.7% and 92.5%. The discriminant function yielded an ARCC 94.8%;
When dog, chicken, cow and pig isolates were pooled together as the animal-group, and all human isolates were combined as the human-group, the ARCC was reduced 89.2%. The RCC was 86.7% for human-group, and 90.1% for animal-group.
The discriminant function analysis among the E.coli isolates of animal-group resulted an ARCC of 96.1%, and 96.7%,94.5%,96.9%and 96.2% of dog, chicken, cow and pig isolates, respectively were identified correctly.
Conclusions
The results of this study have provided evidence about the robustness of rep-PCR DNA fingerprinting analysis in differentiating fecal E.coli strains isolated from humans, and different animals. This method may be possible to accomplish pinpoint identification of specific animal fecal contributions and thereby prevent further pollution.
目的
分析不同粪便来源的大肠杆菌rep-PCR指纹图谱规律,检验rep-PCR方法识别粪便污染源在我国水质监测中是否适用。
方法
本课题大肠杆菌样品采用前期大连海事大学从实验海域、陆源已知污染源分离、生化鉴定纯化后得到的629株大肠杆菌菌株,其中牛源128株,人源159株,鸡源111株,狗源120株,猪源111株；选用E.coli标准菌株ATCC25922为对从样品中分离出的E.coli进行阳性对照。
选择合适的E.coli基因组DNA提取方法。取等量样品,分别用酚／氯仿抽提法、CTAB法改良、CTAB法、液氮法、经典SDS方法、试剂盒法提取大肠杆菌基因组DNA,用紫外分光光度计(260nm,280nm)分别测定其OD值,进行定性、定量分析。确定合适的提取方法,保证有足够DNA模板进行PCR。
rep-PCR反应条件和电泳条件的摸索。对文献中查到的rep-PCR条件进行改良,改变PCR条件、模板加入量、引物浓度等,确定最佳反应条件。
ALPHAVIEW 2.0.0软件数据处理。采用ALPHAVIEW 2.0.0软件分析电泳结果,得到电泳结果图片中条带转化为以数字信息,以DNA marker为标准拟合出一条曲线,以此计算出待测菌株条带DNA片段长度,并以标准菌株的信息为对照验证每次结果的一致性。
指纹图谱分析。按牛、人、鸡、狗、猪不同来源分类,选取5000bp～100bp之间的条带进行分析,分别依照对数曲线确定每段的组距,共分128组,对电泳结果进行分段统计,扩增产物中分子量不同的DNA条带作为多态性状,分子量相同的条带作为一个相同性状,有性状记为1,无性状记为0,总结各来源大肠杆菌rep-PCR共有条带和特征性条带。
对rep-PCR结果进行统计分析。进行聚类分析,建立聚类分析树形图；并分别按照五种来源(牛、人、鸡、狗、猪),两种来源(人、动物),四种来源(牛、鸡、狗、猪)进行判别分析,检验rep-PCR快速鉴别的效果。
结果
考虑细菌基因组DNA提取结果和操作简便性,选择试剂盒法提取样品DNA,为进一步PCR提供足量可靠的模板。
rep-PCR反应体系总结：引物1R(10μM) 2μl、引物21(10μM) 2μl、2×SuperMix 25μl、DNA模板50 ng、加去离子水至50μl；PCR仪反应程序：50μl扩增体系,95℃预变性7 min,94℃变性30s,49.4℃退火1 min,72℃延伸8 min,重复30个循环,最后72℃再延伸16 min。
五种来源大肠杆菌在分子量581～590 bp、1251～1300 bp、3001～3200 bp处存在共有条带,不同种动物粪便中大肠杆菌各自具有特征性条带。
牛源大肠杆菌独有的特征性条带在分子量801～810 bp、1451～1500 bp、2201～2300 bp、2900～3000 bp处；人源大肠杆菌独有的特征性条带在分子量651～660 bp、771～780 bp、1451～1500 bp、2301～2400 bp处；鸡源大肠杆菌独有的特征性条带在分子量601～610bp、691～700bp、2301～2400bp处；狗源大肠杆菌独有的特征性条带在分子量441～450 bp、521～530 bp、771～780 bp、2901～3000 bp处；猪源大肠杆菌独有的特征性条带在分子量511～520 bp、651～660 bp、1451～1500 bp、2501～2600 bp处。
对样品扩增结果进行聚类分析(SPSS 16.0),得到树形图显示分层聚类关系；将样本按照牛、人、鸡、狗、猪不同来源分组,进行判别分析(SPSS 16.0),五组有显著差异(P<0.01),狗源、鸡源、人源、牛源、猪源的RCC值分别为96.7%、98.2%、93.6%、93.7%、92.5%,ARCC值为94.8%；
将四种动物宿主(狗源、鸡源、牛源、猪源)来源样品合并为动物组,人源样品归为一组,两组显示出显著的差异(χ2=440.246,P<0.01),人源RCC为86.7%,动物组RCC为90.1%,ARCC下降到89.2%。
分析四种动物源(狗源、鸡源、牛源、猪源)大肠杆菌图谱,四组有显著差异(P<0.01),狗源、鸡源、牛源、猪源的RCC值分别为96.7%、94.5%、96.9%、96.2%,ARCC值为96.1%。
结论
本课题结果显示：不同粪便来源大肠杆菌rep-PCR产物电泳结果存在共有条带,和各自的特征性条带,并且rep-PCR指纹图谱分析方法可以将在我国海域中采集样品中的大肠杆菌正确分组,能达到污染源示踪的要求,适用于我国水质监测。
For the demand to solve fecal pollution problem in water environment, Microbial Source Tracking (MST), as a new monitoring technology for identifying sources of fecal pollution in water, had emerged in 1990s in US. Using their biochemical characterristics, genetic diversity and its special metabolites to determine their host of the source, thereby identifying fecal pollution sources in water pollution. MST mainly used to deal with the situation that human or animal fecal pollution in the waters increasing and difficult to control, change the non-point pollution into point pollution which can be effective controlled. This subject adopts rep-PCR technology and electrophoresis technology, to qualitative analysis source indicator (E.coli) which collected from different pollution source. Summarizing the fingerprints to test rep-PCR applied to the water quality monitoring in China,in order to provide useful reference to the experts in this field and promote the research and application of MST in our country.
Objective
Summarizing the rep-PCR DNA finger prints analysis of E.coli, and detecting the applicability of rep-PCR method in the water quality monitoring in differentiating fecal pollution source.
Methods
A collection used for this study was collected from experiment maritime space and terrigenous area by researcher of DaLian Maritime University. The collection contained 629 fecal E.coli isolates from humans (159), cow (128), chickens (111), dogs (120) and pigs (111). The study used ATCC25922 as positive control.
To select the best method for extracting the genomic DNA of E.coli. Extracting the genomic DNA of E.coli from equal samples with 5 methods (Phenolic/chloroform, improved CTAB method, CTAB method, the liquid nitrogen, SDS method, and DNA extraction kit method). Using ultraviolet spectrophotometer to measuring OD of DNA templates for qualitative and quantitative analysis. Selecting appropriate methods and ensuring there are adequate DNA template for PCR reaction.
Selecting the best reaction conditions and electrophoresis conditions. Change the conditions of rep-PCR method from literatures, such as PCR conditions, template, concentration of primers for determining the best condition.
Using ALPHAVIEW 2.0.0 software to change the image informations to digital informations. Drawing curve with DNA marker as the standard, to calculate DNA fragment length and use the positive control to test results.
Fingerprint analysis. Dividing the samples into different classifications based on different source (human, cow, chicken, dog and pig). Choosing the bands between 5000bp-100bp for analysis. According to the curve, dividing into 128 groups to analysis the electrophoresis results. Summarizing the common bands and unique bands of rep-PCR fingerprints.
Statistical analysis of rep-PCR DNA fingerprints. The rep-PCR DNA fingerprints were subjected to hierarchical cluster analysis(SPSS 16.0). A dendrogram was constructed by using SPSS. Discriminant function analysis was also performed with the cluster analysis results to find out the number and percentage of isolates from each known source that were classified in each source category.
Results
Considering the genomic DNA results of 5 methods and operation convenience, selecting DNA extraction kit method as DNA extraction method to provide enough sample for PCR reaction.
Rep-PCR mixture:Primers 1R(10μM) 2μl, Primers 21(10μM) 2μl,2×Super Mix 25μl, DNA sample 50ng, up to 50μl with deionized water; Rep-PCR reaction process:95℃for 7 min, and this was followed by 30 cycles consisting of 94℃for 30s,49.4℃for 1 min,72℃for 8 min. The reaction was terminated with an extension step consisting of 72℃for 16 min.
Most isolates'rep-PCR fingerprints contain common bands in molecular weight of 581～590 bp,1251～1300 bp and 3001～3200 bp. Cow isolates'rep-PCR fingerprints contain unique bands in molecular weight of 801-810 bp,1451～1500 bp,2201～2300 bp and 2900～3000 bp; Human isolates'rep-PCR fingerprints contain unique bands in molecular weight of 651-660 bp,771～780 bp,1451～1500 bp and 2301～2400 bp; Chicken isolates'rep-PCR fingerprints contain unique bands in molecular weight of 601～610 bp,691～700 bp and 2301～2400 bp; Dog isolates'rep-PCR fingerprints contain unique bands in molecular weight of 441～450 bp,521～530 bp,771～780 bp and 2901-3000 bp; Pig isolates'rep-PCR fingerprints contain unique bands in molecular weight of 511-520 bp,651～660 bp,1451～1500 bp and 2501～2600 bp.
The cluster analysis was conducted with the rep-PCR DNA fingerprinting patterns of 621 fecal E.coli isolates after removal of the clonal isolates. The dendrogram can not produced obvious clusters. A discriminant function analysis was performed on the cluster analysis results to evaluate how accurately the rep-PCR DNA fingerprints were anle to predict a host source. Dog, chicken, human, cow and pig isolates were highly classified with a RCC of 96.7%,98.2%,93.6%,93.7% and 92.5%. The discriminant function yielded an ARCC 94.8%;
When dog, chicken, cow and pig isolates were pooled together as the animal-group, and all human isolates were combined as the human-group, the ARCC was reduced 89.2%. The RCC was 86.7% for human-group, and 90.1% for animal-group.
The discriminant function analysis among the E.coli isolates of animal-group resulted an ARCC of 96.1%, and 96.7%,94.5%,96.9%and 96.2% of dog, chicken, cow and pig isolates, respectively were identified correctly.
Conclusions
The results of this study have provided evidence about the robustness of rep-PCR DNA fingerprinting analysis in differentiating fecal E.coli strains isolated from humans, and different animals. This method may be possible to accomplish pinpoint identification of specific animal fecal contributions and thereby prevent further pollution.
引文
[1]Szewzyk U, Szewzyk R, Manz W, et al. Microbiological safety of drinking water[J]. Annual Review of Microbiology,2000,54:81-127.
[2]Tao Y, Michael J. Sadowsky. Determining Sources of Fecal Bacteria in Waterways[J]. Environ Monit Assess,2007,129:97-106.
[3]Bernhard AE, and K GF. A PCR assay to discriminate human and animal feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA[J]. Environ. Microbiol,2000,66:4571-4574.
[4]Guan S, R Xu, Chert S, et al. Development of a procedure for discriminating among Escherichia coli isolates from animals and human sources[J]. Environ Microbiol,2002,68:2690-2698.
[5]HaZel PG, Summer JD, Hill JL,et al. Geographic variability of Escherichia coli ribotypes from animals in Idaho and Georgia[J]. Environ Qual,2002,31: 1273-1278.
[6]Joycem S, Jorgew S. Microbial Source Tracking:State of the Scienc[J]. ENVIRONMENTAL SCIENCE & TECHNOLOGY,2002,36:5279-5288.
[7]周早弘,张敏新.防治农业面源污染的工程技术[J].环境与可持续发展,2007,13(6)：55-57.
[8]Guzewich J J, Morse D L. Sources of shellfish in outbreaks of probable viral gastroenteritis:implications for control[J]. Food Prot,1986,49:389-394.
[9]Stelma C N, McCabe L J. Nonpoint pollution from animal sources and shellfish sanitation[J], Food Prot,1992,55:649-656.
[10]贺杰,林凤翱.渤黄海沿岸几种经济贝类及其生存环境中的粪大肠菌群[J].海洋环境科学,2002,21(02)：42-46.
[11]王凯军.禽音养殖污染防治技术与政策[M],化学工业出版社,2004,44-62.
[12]沈国英,施并章.海洋生态学[M].科学出版社,2003,398-399.
[13]王耀兵,苏洁,杨玉敏,等.一种粪便污染源识别新技术—微生物源示踪[J].海洋环境科学,2008,27：122-128.
[14]杨国胜.长江水保所与美国麦普公司建立合作伙伴关系www.ywrt.gov.cn /sbownews.sp/Nes/224,2003,10-28.
[15]Ramaiah N. Marine environmental pollution stress detection throughdirect viable counts of bacteria [J]. Water Research,2002,36:2383-2393.
[16]Whitlock J E, Jones D T. Identification of the sources of fecal coliforms in an urban watershed using antibiotic resistance analysis[J]. WATER RESEARCH, 2002,36(17):4273-4282.
[17]Wheeler A L, Hartel P G, Godffey D G, et al. Potential of Enterococcus faecalis as a Human Fecal Indicator for Microbial Source Tracking[J]. Environ Qual, 2002,31:1286-1293.
[18]U.S. Environmental Protection Agency. Improved enumeration methods for recreational water quality indicators:enterococci and Escherichia coli. EPA/821/R-97/004. Office of Science and Technology, Washington, D.C.2000.
[19]Harwood V J, J Whitlock, V Withington. Classification of antibiotic resistance patterns of indicator bacteria by discriminant analysis:use in predicting the source of fecal contamination in subtropical waters[J]. Environ Microbiol,2000, 66:3698-3704.
[20]Scott T M, Rose J B, Jenkins T M, et al. Microbial source tracking:current methodology and future directions [J]. Applied and Environmental Micro--biology,2002,68(12):5796-5803.
[21]Geldreich E E, Kenner B A. Concepts of fecal streptococci in stream pollution [J]. Water Pollut Control Fed,1969,41:336-352.
[22]Clesceri L S, Greenberg A E, Eaton A D.. Standard methods for the examination of water and wastewater,20th ed American Public Health Association,1998, Washington, D.C.
[23]Pourcher A M, Devriese L A, Hernandez J F, et al. Enumeration by a minia--turized method of E. coli, Streptococcus bovis, and enterococci as indicators of the origin of faecal pollution in waters [J]. Bacteriol,1991,70:525-530.
[24]Cabelli V J, Dufour A P, Levin M A,et al. Swimmingassociated gastroenteritis and water quality[J]. Epidemiol,1982,115:606-616.
[25]Cabelli V J. Health effects for marine recreation waters. USEPA600/1-80-031 Health Effects Research Laboratory,1983. Research Triangle Park, N.C.
[26]Desmarais T R, Solo-Gabriele H M, Palmer C J. Influence of soil on fecal indicator organisms in a tidally influenced subtropical environment[J]. Environ Microbiol,2002,68:1165-1172.
[27]Tartera C, Jofre J. Bacteriophages active against Bacteroides fragilis in sewage-polluted waters[J]. Applied and Environmental Microbiology,1987, 53(7):1632-1637.
[28]Tartera C, Luvcna F, Jofre J. Human origin of Bactemides fragilis bacterio--phages present in the environment[J]. Applied and Environmental Micro--biology,1989,55(10):2696-2701.
[29]Jofre J, Bosch A, Lucena F, et al. Evaluation of Bacteroides fragilis bacterio--phages as indicators of the virological quality of water [J]. Water Science and Technology,1986,18(10):167-173.
[30]Ley V, Higgins J, Fayer R. Bovine enteroviruses as indicators of fecal contamination[J]. Applied and Environmental Microbiology,2002,68(7):3455-3461.
[31]Jimenez C, M A., Fernandez C, et al. Teschoviruses as indicators of porcine fecal contamination of surface water [J]. Applied and Environmental Micro--biology,2003,69(10):6311-6315.
[32]Harwood V J, Whitlock J, Withington V. H. Classification of the antibiotic resistance patterns of indicator bacteria by discriminant analysis:use in predicting the source of fecal contamination in subtropical Florida waters [J]. Environ Microbiol,2000,66:3698-3704.
[33]Wiggins B A. Discriminant analysis of antibiotic resistance patterns in fecal streptococci, a method to differentiate human and animal sources of fecal pollution in natural waters[J]. Environ Microbiol,1996,62:3997-4002.
[34]Wiggins B A, Andrews R A. Conway C L, et al. Use of antibiotic resistance analysis to identify nonpoint sources of fecal pollution[J]. Environ Microbiol, 1999,65:3484-3486.
[35]Hagedorn C S, Robinson S L., Filtz J R.,et al. Using antibiotic resistance patterns in the fecal streptococci to determine sources of fecal pollution in a rural Virginia watershed[J]. Environ Microbiol,1999,65:5522-5531.
[36]Burkhardt M R. Determination of submicrogram-per liter concentrations of caffeine in surface water and groundwater samples by solid-phase extraction and liquid chromatography[J]. AOAC Int,1999,82:161-166.
[37]Leeming R, A Ball, N Ashbolt, et al. Using fecal sterols from humans and animals to distinguish fecal pollution in receiving waters. Water Res,1996, 30:2893-2900.
[38]Hughes J B, Hellmann J J, Ricketts T H et al. Counting the uncountable statistical approaches to estimating microbial diversity[J]. Environ Microbio, 2001,67:4399-4406.
[39]Gordon D M. Geographical structure and host specificity in bacteria and the implications for tracing the source of coliform contamination[J]. Microbiology, 2001,147:1079-1085.
[40]裴德翠等.AFLP:DNA指纹分析的有力手段[J].微生物学免疫学进展,2002,30(3)：66-70.
[41]王玲玲等.AFLP和RAPD标记技术在栉孔扇贝遗传多样性研究中的应用比较[J].动物学杂志,2003,38(4)：35-39.
[42]Kate W, Yutao L, John B. Genetic mapping of the black tiger shrimp Penaeus monodon with amplified fragment length polymorphism[J]. Genetics,2002, 204:297-309.
[43]黄庆生,王加启.16S rRNA/rDNA序列分析技术在瘤胃细菌微生态系统研究中的应用[J].中国畜牧兽医,2003,30(1)：7-11.
[44]Leung K T, Mackereth R, Tien Y C, et al. A comparison of AFLP and ERIC-PCR analyses for discriminating Escherichia coli from cattle, pig and human sources[J]. FEMS MICROBIOLOGY ECOLOGY,2004,47(1):111-119.
[45]Dombek PE, Johnson LK, Zimmerley ST, et al. Use of repetitive DNA sequences and the PCR to differentiate Eseheriehia coli isolates from human and animal sources[J]. APPLIED AND ENVIRONMENTAL MICROBIOLO-GY.2000,66(6):2572-2577.
[46]Carson C A, Shear B L, Ellersieck M R, et al. Identification of fecal Escherichia coli from humans and animals by ribotyping[J]. Environ, Microbiol.2001, 67:1503-1507.
[47]Dombek P E, Johnson L K, Zimmerley, S T, et al. Use of repetitive DNA sequences and the PCR to differentiate Escherichia coli isolates from human and animal sources[J]. Environ Microbiol.2000,66:2572-2577.
[48]Parveen S, M Portier, K Robinson, et al. Discriminant analysis of ribotype profiles of Escherichia coli for differentiating human and nonhuman sources of fecal pollution[J]. Environ Microbiol,1999,65:3142-3147.
[49]Cornuet J, S Pity, G Luikart,et al. New methods employing multilocus geno--types to select or exclude populations as origins of individuals[J]. Genetics, 1999,153:1989-2000.
[50]Chao A. Non-parametric estimation of the number of classes in a population[J]. Scand.J Stat,1984,11:265-270.
[51]Christopher W K, Johanna C A, Mary E S, et al.16S Ribosomal DNA Terminal Restriction Fragment Pattern Analysis of Bacterial Communities in Feces of Rats Fed Lactobacillus acidophilus NCFM[J]. Applied and Environmental Microbiology,2001,67(4):1935-1939.
[52]Nick G, Lindstrom,K. Use of repetitive sequences and the polymerase chain reaction to fingerprint the genomic DNA of Rhizobium galegae strains and to identify the DNA obtained by sonicating the liquid cultures and root nodules[J]. Microbiol,1994,17:265-273.
[53]Buchan A, Alber M, Hodson R E. Strain-specific differentiation of environment--al Eseherichia coli isolates via denaturing gradient gel electrophoresis(DGGE) analysis of the 16S-23S intergenic spacer region[J]. FEMS Microbiol Ecol, 2001,35:313-321.
[54]Stern M J, Ames G F L. Repetive extragenic palindomic sequence:a major component of the bacterial genome[J]. Cell,1984,37:1015-1026.
[55]Versalovic J, Koeuth T, Lupski J R, et al. Distribution of repetitive DNA sequences in eubacteria and application to fingerpringting of bacterial genomes [J]. Nucleic Acid Res,1991,19:6823-6831.
[56]宫强.大肠埃希氏杆菌16S-23S rRNA基因间隔区片断分析在粪便污染源示踪上的应用[D],大连：海事大学2005,1-68.
[57]高定,陈同斌,刘斌.我国畜禽养殖业粪便污染风险与控制策略.地理研究[J].2006,25(2)：311-319.
[58]Wiggins B A, R W Andrews, R A Conway, et al. Use of antibiotic resistance analysis to identify nonpoint sources of fecal pollution[J]. Environ Microbiol, 1999,65:3483-3486.
[59]Parveen S, K M Portier, K Robinson, et al. Discriminant analysis of ribotype profiles of Escherichia coli for differentiating human and nonhuman sources of fecal pollution[J]. Environ Microbiol,1999,65:3142-3147.
[60]Carson C A, Shear B L, Ellersieck M R, et al. Comparison of ribotyping and repetitive extragenic plaindromic-PCR for identification of fecal Escherichia xoli from humans and animals[J]. Environ Mircrobiol.2003.69:1836-1839.
[61]何亮,陈群,曾忠铭等.通过特异PCR扩增和16SrDNA序列分析检测动弯杆菌[J].微生物学报,2005,41：27-30.
[62]Suaua A, Bonnet R,Sutren M, et al. Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut[J]. Applied Enviro Microbiol,1999,65(11):4799-4807.
[63]Duda R O, P E Hart, D G Stork. Pattern classification,2nd ed. John Wiley and Sons, Inc. New York.2001.
[64]Mehta A, Mehta R,Tosato Y B. ERIC and REP-PCR amplify non-repetitive fragments from the genome of Drechslera avenae and Stremphylium solani[J]. FEMS Microbiology Letters,2002,211:21-55.
[65]Takeshi T Mitsuro H.Genetic relatedness among and within different Rhizocto--nia solani anastomosis groups as assessde by RAPD,ERIC and REP-PCR[J]. Microbiol Res,1999,154(3):247-258.
[66]Del V G, Petroziellom J, Gress M J,et al.Molecular genotypeing of methicillin resistant Staphylococcus aureus via fluorophore enhanc-ed repetitive sequence PCR[J]. J Clin Microbiol,1995,33(8):2141-2144.
[67]Vander Z A, Verbakel H, Vanzon J C,et al.Molecular genotyping of Staphylo--coccus aureus stains:comparision of repetitive element sequence based PCR with various typing methods and isolation of a novelepidemicity marker [J].J Clin Microbiol,1999,37(2):342-349.
[68]Xufre A, Simoes F,Girio F, et al. Use of RAPD analysis for differentiation among six enological Saccharomyces strains [J]. Food Technol Biotechnd,2000, 38(1):53-58.
[69]Jurkovic D, Krizkova L, Sojka M, et al. Genetic diversity of Entero--coccus faecium isolated from Bryndza cheese [J]. International Journal of Food Microbiology,2007,116:82-87.
[70]Borgesl D A, Vechia N D, Corca G. Characterisation and genetic diversity via REP-PCR of Staphylococcus aureus isolates from polluted waters in southern Brazil[J]. FEMS Microbiology Ecology,2003,45:173-180.
[71]Ibenyassine K, Aitmhand R, Karamoko Y, et al. Use of repetitive DNA sequ-ences to determine the persistence of enteropathogenic Escherichia coli in vegetables and in soil grown in fields treated with contaminated irrigation water [J]. Letters in Spplide Microbiology,2006,43:528-533.
[72]Olive D M, Bean P. Principles and applications of methods for DNA based typing of microbial organisms[J]. Journal of Clinical Microbiology,1999,37(6): 1661-1669.
[73]Dombek P E, Johnson L K, Zimmerley S T, et al. Use of repetitive DNA sequences and the PCR to differentiate Escherichia coli isolates from human and animal sources[J]. Environ. Microbiol,2000,66:2572-2577.
[74]Guan S, Xu R, Chen S, et al. Development of a procedure for discriminating among Escherichia coli isolates from animal and human sources[J]. Environ Microbiol,2007,68:2690-2698.
[75]Mohapatra B R, Broersma K, Nordin B, et al. Evaluation of repetitive extra--genic palindromic-PCR for discrimination of fecal Escherichia coli from humans, and different domestic-and wild-animals [J]. Microbiol Immunol,2007, 51(8):733-740.
[76]Seurinck S., Verstraete W., Siciliano S D. Use of 16S-23S rRNA intergenic spacer region PCR and repetitive extragenic palindromic PCR analyses of Escherichia coli isolates to identify nonpoint fecal sources[J]. Environ Microbiol,2003,69:4942-4950.
[77]Somarelli J A., Makarewicz J C, Sia R. Wildlife identified as major source of Escherichia coli in agriculturally dominated watersheds by BOX AlR-derived genetic fingerprints [J]. Environ Manage,2007,82:60-65.
[78]Hartel P G., Summer J D Segars W I. Deer diet affects ribotype diversity of Escherichia coli for bacterial source tracking. Water Res,2003,37:3263-3268.
[79]Johnson L K, Brown M B., Carruthers E A, et al. Sample size, library composi--tion, and genotypic diver among natural populations of Escherichia coli from different animals influence accuracy of determining sources of fecal pollution [J]. Environ Microbiol,2004,70:4478-4485.
[80]Harwood V J, Whitlock J, WithingtonV. Classification of antibiotic resistance patterns of indicator bacteria by discriminant analysis:use in predicting the source of fecal contamination in subtropical waters[J]. Environ Microbiol,2000, 66:3698-3704.
[2]Tao Y, Michael J. Sadowsky. Determining Sources of Fecal Bacteria in Waterways[J]. Environ Monit Assess,2007,129:97-106.
[3]Bernhard AE, and K GF. A PCR assay to discriminate human and animal feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA[J]. Environ. Microbiol,2000,66:4571-4574.
[4]Guan S, R Xu, Chert S, et al. Development of a procedure for discriminating among Escherichia coli isolates from animals and human sources[J]. Environ Microbiol,2002,68:2690-2698.
[5]HaZel PG, Summer JD, Hill JL,et al. Geographic variability of Escherichia coli ribotypes from animals in Idaho and Georgia[J]. Environ Qual,2002,31: 1273-1278.
[6]Joycem S, Jorgew S. Microbial Source Tracking:State of the Scienc[J]. ENVIRONMENTAL SCIENCE & TECHNOLOGY,2002,36:5279-5288.
[7]周早弘,张敏新.防治农业面源污染的工程技术[J].环境与可持续发展,2007,13(6)：55-57.
[8]Guzewich J J, Morse D L. Sources of shellfish in outbreaks of probable viral gastroenteritis:implications for control[J]. Food Prot,1986,49:389-394.
[9]Stelma C N, McCabe L J. Nonpoint pollution from animal sources and shellfish sanitation[J], Food Prot,1992,55:649-656.
[10]贺杰,林凤翱.渤黄海沿岸几种经济贝类及其生存环境中的粪大肠菌群[J].海洋环境科学,2002,21(02)：42-46.
[11]王凯军.禽音养殖污染防治技术与政策[M],化学工业出版社,2004,44-62.
[12]沈国英,施并章.海洋生态学[M].科学出版社,2003,398-399.
[13]王耀兵,苏洁,杨玉敏,等.一种粪便污染源识别新技术—微生物源示踪[J].海洋环境科学,2008,27：122-128.
[14]杨国胜.长江水保所与美国麦普公司建立合作伙伴关系www.ywrt.gov.cn /sbownews.sp/Nes/224,2003,10-28.
[15]Ramaiah N. Marine environmental pollution stress detection throughdirect viable counts of bacteria [J]. Water Research,2002,36:2383-2393.
[16]Whitlock J E, Jones D T. Identification of the sources of fecal coliforms in an urban watershed using antibiotic resistance analysis[J]. WATER RESEARCH, 2002,36(17):4273-4282.
[17]Wheeler A L, Hartel P G, Godffey D G, et al. Potential of Enterococcus faecalis as a Human Fecal Indicator for Microbial Source Tracking[J]. Environ Qual, 2002,31:1286-1293.
[18]U.S. Environmental Protection Agency. Improved enumeration methods for recreational water quality indicators:enterococci and Escherichia coli. EPA/821/R-97/004. Office of Science and Technology, Washington, D.C.2000.
[19]Harwood V J, J Whitlock, V Withington. Classification of antibiotic resistance patterns of indicator bacteria by discriminant analysis:use in predicting the source of fecal contamination in subtropical waters[J]. Environ Microbiol,2000, 66:3698-3704.
[20]Scott T M, Rose J B, Jenkins T M, et al. Microbial source tracking:current methodology and future directions [J]. Applied and Environmental Micro--biology,2002,68(12):5796-5803.
[21]Geldreich E E, Kenner B A. Concepts of fecal streptococci in stream pollution [J]. Water Pollut Control Fed,1969,41:336-352.
[22]Clesceri L S, Greenberg A E, Eaton A D.. Standard methods for the examination of water and wastewater,20th ed American Public Health Association,1998, Washington, D.C.
[23]Pourcher A M, Devriese L A, Hernandez J F, et al. Enumeration by a minia--turized method of E. coli, Streptococcus bovis, and enterococci as indicators of the origin of faecal pollution in waters [J]. Bacteriol,1991,70:525-530.
[24]Cabelli V J, Dufour A P, Levin M A,et al. Swimmingassociated gastroenteritis and water quality[J]. Epidemiol,1982,115:606-616.
[25]Cabelli V J. Health effects for marine recreation waters. USEPA600/1-80-031 Health Effects Research Laboratory,1983. Research Triangle Park, N.C.
[26]Desmarais T R, Solo-Gabriele H M, Palmer C J. Influence of soil on fecal indicator organisms in a tidally influenced subtropical environment[J]. Environ Microbiol,2002,68:1165-1172.
[27]Tartera C, Jofre J. Bacteriophages active against Bacteroides fragilis in sewage-polluted waters[J]. Applied and Environmental Microbiology,1987, 53(7):1632-1637.
[28]Tartera C, Luvcna F, Jofre J. Human origin of Bactemides fragilis bacterio--phages present in the environment[J]. Applied and Environmental Micro--biology,1989,55(10):2696-2701.
[29]Jofre J, Bosch A, Lucena F, et al. Evaluation of Bacteroides fragilis bacterio--phages as indicators of the virological quality of water [J]. Water Science and Technology,1986,18(10):167-173.
[30]Ley V, Higgins J, Fayer R. Bovine enteroviruses as indicators of fecal contamination[J]. Applied and Environmental Microbiology,2002,68(7):3455-3461.
[31]Jimenez C, M A., Fernandez C, et al. Teschoviruses as indicators of porcine fecal contamination of surface water [J]. Applied and Environmental Micro--biology,2003,69(10):6311-6315.
[32]Harwood V J, Whitlock J, Withington V. H. Classification of the antibiotic resistance patterns of indicator bacteria by discriminant analysis:use in predicting the source of fecal contamination in subtropical Florida waters [J]. Environ Microbiol,2000,66:3698-3704.
[33]Wiggins B A. Discriminant analysis of antibiotic resistance patterns in fecal streptococci, a method to differentiate human and animal sources of fecal pollution in natural waters[J]. Environ Microbiol,1996,62:3997-4002.
[34]Wiggins B A, Andrews R A. Conway C L, et al. Use of antibiotic resistance analysis to identify nonpoint sources of fecal pollution[J]. Environ Microbiol, 1999,65:3484-3486.
[35]Hagedorn C S, Robinson S L., Filtz J R.,et al. Using antibiotic resistance patterns in the fecal streptococci to determine sources of fecal pollution in a rural Virginia watershed[J]. Environ Microbiol,1999,65:5522-5531.
[36]Burkhardt M R. Determination of submicrogram-per liter concentrations of caffeine in surface water and groundwater samples by solid-phase extraction and liquid chromatography[J]. AOAC Int,1999,82:161-166.
[37]Leeming R, A Ball, N Ashbolt, et al. Using fecal sterols from humans and animals to distinguish fecal pollution in receiving waters. Water Res,1996, 30:2893-2900.
[38]Hughes J B, Hellmann J J, Ricketts T H et al. Counting the uncountable statistical approaches to estimating microbial diversity[J]. Environ Microbio, 2001,67:4399-4406.
[39]Gordon D M. Geographical structure and host specificity in bacteria and the implications for tracing the source of coliform contamination[J]. Microbiology, 2001,147:1079-1085.
[40]裴德翠等.AFLP:DNA指纹分析的有力手段[J].微生物学免疫学进展,2002,30(3)：66-70.
[41]王玲玲等.AFLP和RAPD标记技术在栉孔扇贝遗传多样性研究中的应用比较[J].动物学杂志,2003,38(4)：35-39.
[42]Kate W, Yutao L, John B. Genetic mapping of the black tiger shrimp Penaeus monodon with amplified fragment length polymorphism[J]. Genetics,2002, 204:297-309.
[43]黄庆生,王加启.16S rRNA/rDNA序列分析技术在瘤胃细菌微生态系统研究中的应用[J].中国畜牧兽医,2003,30(1)：7-11.
[44]Leung K T, Mackereth R, Tien Y C, et al. A comparison of AFLP and ERIC-PCR analyses for discriminating Escherichia coli from cattle, pig and human sources[J]. FEMS MICROBIOLOGY ECOLOGY,2004,47(1):111-119.
[45]Dombek PE, Johnson LK, Zimmerley ST, et al. Use of repetitive DNA sequences and the PCR to differentiate Eseheriehia coli isolates from human and animal sources[J]. APPLIED AND ENVIRONMENTAL MICROBIOLO-GY.2000,66(6):2572-2577.
[46]Carson C A, Shear B L, Ellersieck M R, et al. Identification of fecal Escherichia coli from humans and animals by ribotyping[J]. Environ, Microbiol.2001, 67:1503-1507.
[47]Dombek P E, Johnson L K, Zimmerley, S T, et al. Use of repetitive DNA sequences and the PCR to differentiate Escherichia coli isolates from human and animal sources[J]. Environ Microbiol.2000,66:2572-2577.
[48]Parveen S, M Portier, K Robinson, et al. Discriminant analysis of ribotype profiles of Escherichia coli for differentiating human and nonhuman sources of fecal pollution[J]. Environ Microbiol,1999,65:3142-3147.
[49]Cornuet J, S Pity, G Luikart,et al. New methods employing multilocus geno--types to select or exclude populations as origins of individuals[J]. Genetics, 1999,153:1989-2000.
[50]Chao A. Non-parametric estimation of the number of classes in a population[J]. Scand.J Stat,1984,11:265-270.
[51]Christopher W K, Johanna C A, Mary E S, et al.16S Ribosomal DNA Terminal Restriction Fragment Pattern Analysis of Bacterial Communities in Feces of Rats Fed Lactobacillus acidophilus NCFM[J]. Applied and Environmental Microbiology,2001,67(4):1935-1939.
[52]Nick G, Lindstrom,K. Use of repetitive sequences and the polymerase chain reaction to fingerprint the genomic DNA of Rhizobium galegae strains and to identify the DNA obtained by sonicating the liquid cultures and root nodules[J]. Microbiol,1994,17:265-273.
[53]Buchan A, Alber M, Hodson R E. Strain-specific differentiation of environment--al Eseherichia coli isolates via denaturing gradient gel electrophoresis(DGGE) analysis of the 16S-23S intergenic spacer region[J]. FEMS Microbiol Ecol, 2001,35:313-321.
[54]Stern M J, Ames G F L. Repetive extragenic palindomic sequence:a major component of the bacterial genome[J]. Cell,1984,37:1015-1026.
[55]Versalovic J, Koeuth T, Lupski J R, et al. Distribution of repetitive DNA sequences in eubacteria and application to fingerpringting of bacterial genomes [J]. Nucleic Acid Res,1991,19:6823-6831.
[56]宫强.大肠埃希氏杆菌16S-23S rRNA基因间隔区片断分析在粪便污染源示踪上的应用[D],大连：海事大学2005,1-68.
[57]高定,陈同斌,刘斌.我国畜禽养殖业粪便污染风险与控制策略.地理研究[J].2006,25(2)：311-319.
[58]Wiggins B A, R W Andrews, R A Conway, et al. Use of antibiotic resistance analysis to identify nonpoint sources of fecal pollution[J]. Environ Microbiol, 1999,65:3483-3486.
[59]Parveen S, K M Portier, K Robinson, et al. Discriminant analysis of ribotype profiles of Escherichia coli for differentiating human and nonhuman sources of fecal pollution[J]. Environ Microbiol,1999,65:3142-3147.
[60]Carson C A, Shear B L, Ellersieck M R, et al. Comparison of ribotyping and repetitive extragenic plaindromic-PCR for identification of fecal Escherichia xoli from humans and animals[J]. Environ Mircrobiol.2003.69:1836-1839.
[61]何亮,陈群,曾忠铭等.通过特异PCR扩增和16SrDNA序列分析检测动弯杆菌[J].微生物学报,2005,41：27-30.
[62]Suaua A, Bonnet R,Sutren M, et al. Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut[J]. Applied Enviro Microbiol,1999,65(11):4799-4807.
[63]Duda R O, P E Hart, D G Stork. Pattern classification,2nd ed. John Wiley and Sons, Inc. New York.2001.
[64]Mehta A, Mehta R,Tosato Y B. ERIC and REP-PCR amplify non-repetitive fragments from the genome of Drechslera avenae and Stremphylium solani[J]. FEMS Microbiology Letters,2002,211:21-55.
[65]Takeshi T Mitsuro H.Genetic relatedness among and within different Rhizocto--nia solani anastomosis groups as assessde by RAPD,ERIC and REP-PCR[J]. Microbiol Res,1999,154(3):247-258.
[66]Del V G, Petroziellom J, Gress M J,et al.Molecular genotypeing of methicillin resistant Staphylococcus aureus via fluorophore enhanc-ed repetitive sequence PCR[J]. J Clin Microbiol,1995,33(8):2141-2144.
[67]Vander Z A, Verbakel H, Vanzon J C,et al.Molecular genotyping of Staphylo--coccus aureus stains:comparision of repetitive element sequence based PCR with various typing methods and isolation of a novelepidemicity marker [J].J Clin Microbiol,1999,37(2):342-349.
[68]Xufre A, Simoes F,Girio F, et al. Use of RAPD analysis for differentiation among six enological Saccharomyces strains [J]. Food Technol Biotechnd,2000, 38(1):53-58.
[69]Jurkovic D, Krizkova L, Sojka M, et al. Genetic diversity of Entero--coccus faecium isolated from Bryndza cheese [J]. International Journal of Food Microbiology,2007,116:82-87.
[70]Borgesl D A, Vechia N D, Corca G. Characterisation and genetic diversity via REP-PCR of Staphylococcus aureus isolates from polluted waters in southern Brazil[J]. FEMS Microbiology Ecology,2003,45:173-180.
[71]Ibenyassine K, Aitmhand R, Karamoko Y, et al. Use of repetitive DNA sequ-ences to determine the persistence of enteropathogenic Escherichia coli in vegetables and in soil grown in fields treated with contaminated irrigation water [J]. Letters in Spplide Microbiology,2006,43:528-533.
[72]Olive D M, Bean P. Principles and applications of methods for DNA based typing of microbial organisms[J]. Journal of Clinical Microbiology,1999,37(6): 1661-1669.
[73]Dombek P E, Johnson L K, Zimmerley S T, et al. Use of repetitive DNA sequences and the PCR to differentiate Escherichia coli isolates from human and animal sources[J]. Environ. Microbiol,2000,66:2572-2577.
[74]Guan S, Xu R, Chen S, et al. Development of a procedure for discriminating among Escherichia coli isolates from animal and human sources[J]. Environ Microbiol,2007,68:2690-2698.
[75]Mohapatra B R, Broersma K, Nordin B, et al. Evaluation of repetitive extra--genic palindromic-PCR for discrimination of fecal Escherichia coli from humans, and different domestic-and wild-animals [J]. Microbiol Immunol,2007, 51(8):733-740.
[76]Seurinck S., Verstraete W., Siciliano S D. Use of 16S-23S rRNA intergenic spacer region PCR and repetitive extragenic palindromic PCR analyses of Escherichia coli isolates to identify nonpoint fecal sources[J]. Environ Microbiol,2003,69:4942-4950.
[77]Somarelli J A., Makarewicz J C, Sia R. Wildlife identified as major source of Escherichia coli in agriculturally dominated watersheds by BOX AlR-derived genetic fingerprints [J]. Environ Manage,2007,82:60-65.
[78]Hartel P G., Summer J D Segars W I. Deer diet affects ribotype diversity of Escherichia coli for bacterial source tracking. Water Res,2003,37:3263-3268.
[79]Johnson L K, Brown M B., Carruthers E A, et al. Sample size, library composi--tion, and genotypic diver among natural populations of Escherichia coli from different animals influence accuracy of determining sources of fecal pollution [J]. Environ Microbiol,2004,70:4478-4485.
[80]Harwood V J, Whitlock J, WithingtonV. Classification of antibiotic resistance patterns of indicator bacteria by discriminant analysis:use in predicting the source of fecal contamination in subtropical waters[J]. Environ Microbiol,2000, 66:3698-3704.