Comparison And Application Of Two Different Kinds Of Microbial Source Tracking Techniques

Microbial Source Tracking (MST) is a new technique basing on the assumption that by monitoring the microbial indicators with molecular and biochemical methods, the origin of pathogenic microorganisms can be tracked and controlled. MST can also assess the contributions of the various contamination sources. It is supposed to be an effective approach to control non-point fecal contamination. In this issue, two library-dependent and library independent methods are compared and applied. This study can provide a theoretical basis for further research and application reference for monitoring and managing of non-point source fecal pollution.ObjectThe fecal samples of different sources were collected from Dalian and Jinan. By tracking the pollution sources with library-dependent and library-independent microbial source tracking methods, the accuracy and stability of these two kinds of methods, especially the impact of geographic variation were compared. The application value of microbial source tracking was analysed by tracking the contamination sources of water samples with both of library-dependent and library-independent methods and the contribution of different contamination sources were assessed.MethodsDalian and Jinan were set as sampling points. Water samples and fecal samples from chicken, dog, cattle, pig and human sources were collected by sterile operation. The ATCC25922were used as standard E.coli in this issue. Eosin methylene blue (EMB) tests, sugar fermentation tests, IMVIC tests were used to isolate and identify E.coli from the samples. DNA were extracted from E.coli samples by EasyPure Genomic DNA Extraction Kit and tested by UV spectrophotometer. OD600, OD260and OD280were used to test whether the DNA is pure enough to be used for PCR. Gel image analysis system was used to obtain the DNA fingerprints which were produced by REP-PCR and Agarose gel electrophoresis. By ALPHAVIEW software, the picture of DNA fingerprints were translated into digital data, including position, size, relative brightness, grayscale, and area information. The SPSS software was used to analysis the digital DNA fingerprints and perform source tracking with library-dependent and library-independent methods. The Rates of Correct Classification (RCC) of different areas, different sources and different methods were compared. The microbial source tracking was also performed on E.coli isolates which were obtained from water samples.ResultsTotally380fecal samples were collected, in which,210were from Dalian and170were from Jinan. Totally15water samples were collected from Jinan.1171E.coli isolates were obtained by isolation and identification (616from Dalian,555from Jinan). The DNA was extracted by EasyPure Genomic DNA Extraction Kit. By REP-PCR,1171products were obtained. From the DNA fingerprints, we could see the bands located from100bp to5000bp, especially the area from500bp to2000bp. Isolates from the same source shared more specific bands. Though isolates from the same host source and different areas shown certain variation, they still shared some specific bands. The repeated DNA fingerprints were removed from the database to prevent bias. Totally889isolates were left including500isolates from Dalian and389isolates from Jinan. The database was then analyzed by ALPHAVIEW and SPSS software.Track the source of500E.coli isolates from Dalian fecal sample by library-dependent and library-independent methods. The RCC of library-dependent method is chicken-94.9%, dog-95%, cattle-99.1%, pig-95%and human-95.1%and the ARCC is95.8%. The ARCC of library-independent method is chicken-94.9%, dog-95%, cattle-99.1%, pig-95%and human-95.1%and the ARCC is95.8%.Track the source of389E.coli isolates from Jinan fecal sample by library-dependent and library-independent methods. The RCC of library-dependent method is chicken-98.5%, dog-97.1%, cattle-100%, pig-96.9%and human-98.3%and the ARCC is98.2%. The ARCC of library-independent method is chicken-78.8%, dog-50%, cattle-75.3%, pig-71.9%and human-62.1%and the ARCC is66.8%.Track the source of889E.coli isolates from fecal sample by library-dependent and library-independent methods. The RCC of library-dependent method is Dalian-chicken92.4%, Dalian-dog90.0%, Dalian-cattle88.5%, Dalian-pig89.3%and Dalian-human85.2%, Jinan-chicken87.9%, Jinan-dog92.9%, Jinan-cattle97.3%, Jinan-pig97.5%and Jinan-human90.5%. The ARCC is89.9%. The RCC of library-independent method is Dalian-chicken60.5%, Dalian-dog48.0%, Dalian-cattle58.4%, Dalian-pig12.3%and Dalian-human23.8%, Jinan-chicken74.7%, Jinan-dog41.4%, Jinan-cattle56.2%, Jinan-pig65.6%and Jinan-human57.8%. The ARCC is47.7%.Track the source of E.coli isolates from water sample by library-dependent and library-independent methods. The results show that though the main source of non-point fecal pollution is unkown, human source, chicken source and dog source contribute a large part of non-point fecal pollution.ConclusionThe results show that REP-PCR based MST is reliable. Most of the RCCs and ARCCs by two methods are higher. The library-dependent MST performs more accurate and steady:higher than94%on same area and higher85%on different areas, which is significantly higher than library-independent method (P<0.05). Especially the stability on geographic variation implies the possible value of large area application. Meanwhile, the source tracking results show that the main non-point fecal source of Huanhe may be human, chicken and dog though a large portion is still unknown. These results provide a good reference to future study and application of microbial source tracking.