| 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.ObjectiveSummarizing 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.MethodsA 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.ResultsConsidering 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.ConclusionsThe 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.
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