The Research Of Detection Duck Fecal Pollution In Environment Water Using Faecalibacterium 16S RDNA Genetic Marker

With the rapid growth of livestock and poultry food demand,the livestock and poultry breeding industry has also developed rapidly.However,the problem of animal manure pollution caused by large-scale farming cannot be neglected.And the feces contain a large number of pathogenic bacteria,which pose a serious threat to the ecological environment and human health.Therefore,the technology to accurately and quickly detect the source of water faecal contamination is essential.However,traditional fecal indicator bacteria(such as Escherichia coli,Enterococcus,etc.)can survive and reproduce in a suitable environment outside the host,and it is difficult to truly reflect the situation of water pollution.In this study,Faecalibacterium was selected as a faecal indicator.According to the sequence of 16S rDNA gene of Faecalibacterium among different species,duck-specific genetic marker was designed to detect duck faecal contamination in water.A total of 246 stool samples from six common species of human,duck,chicken,pig,dog and cattle were collected in this experiment.646,587 effective 16S rDNA sequences were obtained through high-throughput sequencing,and the microbial diversity of individual faecal was analyzed.The results showed that the abundances of Firmicutes,Bacteroidetes and Proteobacteria were the highest.Firmicutes accounted for 35.45%-68.68%of different animals,Bacteroidetes accounted for 12.86%-47.3%and Proteobacteria accounted for 0.71%-34.41%.Average Shannon index in human,duck,chicken,pig,dog and cattle faecal were 0.9392,1.2174,1.2148,0.8324,1.1547,1.2293,respectively.Average Simpson index were 0.5529,0.6321,0.6257,0.4591,0.5418,0.6422,respectively.Average Pielou index were 0.4241,0.3652,0.3893,0.3397,0.3966,0.4419,respectively.However,the average Shannon index of duck feces in Chongqing,Guizhou,Shaanxi and Sichuan were 1.2217,1.2473,1.2151,1.1869,average Simpson index were 0.6431,0.6647,0.6358,0.6032,and average Pilu index were 0.3772,0.3863,0.3744 and 0.3512,respectively.The specificity of two groups of duck-specific Faecalibacterium primers(FDu-1and FDu-2)designed in this study were 100%.The positive rates were 93.75%and90.63%,respectively.The sensitivity were 7.95×10~7 copies/100 mL and 1.89×10~9copies/100 mL,respectively.Because the positive rate and sensitivity of FDu-1 were higher than FDu-2,FDu-1 was determined as the best primer.In order to evaluate the persistence of FDu-1 in water environment,we used laboratory simulation duck fecal to pollute the water,and selected different water quality(river water,distilled water),different temperature(10°C,30°C),light(light,darkness)as variables.The results showed that under 10°C and dark conditions,the decay time in river water and distilled water of FDu-1 were 108 h and 132 h,respectively.Under 30°C and dark conditions,the decay time in river water and distilled water of FDu-1 were 108 h and 132 h,respectively.Under 10°C and 30°C,the decay time in river water of FDu-1 were 96 h and 36 h,respectively.Finally,to verify the feasibility of FDu-1 in practical use,the environmental water samples were randomly collected from different areas.The results showed that the water samples polluted by duck faecal were positive,while the water samples polluted by non-target faecal were negative.In addition,the farther the distance from the duck faecal sampling point,the lower the positive rate.The duck-specific Faecalibacterium gene marker designed and validated in this paper can independently detect and track duck faecal contamination in water,providing a new tool and method for the detection of faecal contamination sources in water environment.