| Fecal contamination has become more and more serious as the development of swine industry, posing threat to environment and human health. Traditional fecal indicator such as Escherichia coli and Enterococcus can survive and propagate in environmental water, thus are not suitable for tracing pollution source. 126 fecal samples from six species(including chicken, duck, human, dog, pig and cow) were collected and high-energy sequencing technology was used to analyze microbial diversity. Microbial source tracing technology(MST) was used in this study and Faecalibacterium was chosen as swine fecal contamination indicator. Swine feces-specific Faecalibacterium 16 S r DNA sequence was identified and primers were designed based on the sequence identified. Real-time PCR assay was then conducted to detect and trace swine fecal contamination.146, 038 bacterial 16 S r DNA sequences were obtained through high-throughput sequencing. The most abundant phyla in animal feces are Firmicutes, with abundance ranges from 39.10 % to 69.44 %. Followed by Bacteroidetes(10.64 %- 48.41 %) and Proteobacteria(0.060 %- 39.75 %). Average Shannon index were 1.21, 1.22, 1.13, 0.83 and 0.94 for chicken, duck, caw, pig and human, respectively. Average Simpson index were 0.65, 0.63, 0.60, 0.46, and 0.55. A universal primer(FUP) was designed by comparing the conservative and variable region of Faecalibacterium 16 S rDNA from different host species. And two swine feces-specific primers(PFB-1 and PFB-2) were screened. PCR result shows that primer FUP can amplify a target fragment of 237 bp in all the fecal DNA samples. PFB-1 and PFB-2 can only amplify target fragment in swine fecal sample, with product of 172 bp and 225 bp respectively. Both of the two primers were 100 % specific for swine fecal sample, having no cross- reaction with other species. The positive rate of PFB-2(90 %) was higher than PFB-1(86 %). The limits of quantification(LOQs) is 6.5 copies/ 100 ml and 2.9 copies/100 ml for primer PFB-1 and PFB-2 respectively, indicating that primer PFB-2 is more sensitive than primer PFB-1.Thus primer PFB-2 is more suitable for the detection and quantification of swine feces contamination in water bodies. In addition, we simulated swine feces contaminated pond water to analyze the decay characteristic of Faecalibacterium gene marker(PFB-2) under different temperature and illumination using real-time PCR. Under the condition of 15 ° C and darkness, the decay rate is 0.0647 h-1, duration of 71.17 h; At 25 ° C and illumination condition, the decay rate is 0.1112 h-1, duration of 41.41 h, showed that the decay rate of molecular markers were slower under low temperature and dark environment. Finally, environmental water samples were randomly collected and real-time PCR assay was conducted to verify the application of PFB-2 as gene marker for swine fecal contamination. For 3 water samples collected near swine farm, we detected 8.4 ± 0.7×104, 4.5 ± 0.5×10 and 3.6 ± 1.2×102 gene copies respectively. While for samples from other positions, the PCR results were negative. This study provides fast and efficient method for the detection and tracing of swine fecal contamination in water, which can help to ensure water quality. |
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