| Waterborne disease has a great health risk to human, and the main reason contribute pathogens to contaminated water is fecal pollution. Identification of sources of fecal contamination is vital for health hazard evaluation and polluted water management. Traditional methods can only evaluate water quality, but cannot find the sources of pollution. The rep-PCR fingerprinting analysis is one microbial source tracking method, which can identify fecal sources by differentiate gene fingerprinting of different sources combining the statistical analysis.OBJECTIVEThis study developed a known-sources fingerprinting library containing all kinds of animals whose fecal had the chances to pollute the Guiwu reservoir. Then, a library self-cross was used to calculate rates of correct classification to evaluate the method. At last, it was applied to analysis a reservoir samples to identify the fecal pollution sources.METHODSFresh fecal samples,representing human and animals (poultry,domestic animals and wild animals), were collected throughout villages around the reservoir in spring, summer, autumn and winter of a year. Escherichia coli were identified as indicators and PCR was expanded with the support of BOXA1R premier. The rep-PCR fingerprinting was puted into Bionumerics 4.0 software to build a known-sources character fingerprinting library. The rates of correct assignment by jackknife analysis, Multivariate analysis of variance (MANOVA) and discriminant analysis were conducted by software. Water samples were collected once a month and Escherichia coli were identified by membrane filtration, rep-PCR amplification to get fingerprinting, each of which was analyzed by contrasting with the known source library to find it source and the result of total was applied to identify the main fecal pollutions.RESULTS1. Source DNA fingerprints of E.coli were isolated from 650 fecal samples, unique E.coli fingerprints were distinguished from the 2705 fingerprints to build the known-source database.2. The known source fingerprint library which was classified as 10, 5, 3, and 2 groups, the average rates of correct classification were respectively 68.13%, 74.76%, 82.36% and 86.03%. MANOVA and discriminant analysis showed that the division of different groups was clearly. E.coli colonies of unknown source were isolated from 84 water samples taken from six watersheds.3. 502 E.coli fingerprints were selected from the 534 isolates to identify their sources, that 477 E.coli fingerprints can identify their sources, which contain 95.02% of total fingerprints. The result showed that the reservoir's fecal pollution was complex. Human, chicken, cattle, sheep, pigs, ducks, goose, wild animals, dogs, and pigeons respectively account for 27.88%, 15.51 %, 11.53%, 9.22%, 8.39%, 8.18%, 6.01%, 5.66%, 4.19% and 3.35%。Human, chicken and cattle altogether occupied 54.92%。CONCLUSIONThe rep-PCR DNA fingerprint analysis was a promising method to determine the source groups of indictor isolates in water. Management practices should intend to decrease the human, poultry and domestic animals fecal pollutions in Guiwu reservoir, more actions should be done to make non-hazard treatment of feces. KEY WORDS: Microbial source tracking; Rep-PCR; BOXA1R; Escherichia coli. |
PDF Full Text Request