The Research Of Detection Fecal Pollution Sources In Water By Using Cattle Intestinal Faecalibacterium 16S RDNA Genetic

With the development of cattle breeding industry,the excrement of cattle manure has become more and more serious.It has seriously affected and endangered human and animal health and ecosystem balance.However,traditional fecal indicators,such as Escherichia coli and Enterococcus,can propagate and proliferate in an in vitro environment,making it difficult to detect and detect real fecal contamination sources.In this project,Faecalibacterium was selected as the indicator of stool.According to the sequence of 16S rDNA gene of Faecalibacterium,different hosts will show sequence differences to find the specific marker of cattle species.This technique can ensure the accuracy of stool contamination detection.A total of 324 fecal samples from chicken,duck,dog,human,pig and cattle(the most contaminated feces samples)were collected from the feces samples by high-throughput sequencing.High-throughput sequencing obtained a total of 564,224effective 16S rDNA sequences.After data analysis,among the six species of stool samples,the most dominant bacteria were Firmicutes,Bacteroidetes and Proteobacteria,and the proportion of Firmicutes in different animals accounted for 39.01%-69.44%.Bacteroidetes accounted for 12.43%-47.41%,Proteobacteria accounted for about0.79%-38.82%.The Shannon-wiener average indexes of microorganisms in chicken,duck,dog,human,pig and cattle samples were 1.20,1.23,1.16,0.87,0.94,1.30,followed by Simpson average index were 0.65,0.64,0.64,0.59,0.46,0.66,The Pielou average index followed by 0.40,0.37,0.40,0.42,0.35,0.44.However,in cattle species,the abundance of yellow cattle(1.30,0.67)and Pielou index(0.45)and buffalo abundance index(1.31,0.69)and Pielou index(0.45)were close,and all three indexes were higher than that of cow's abundance index(1.23,0.48)and Pielou index(0.43).This result is consistent with our suggestion that buffalo and yellow cattle genomic DNA concentrations are slightly higher than that of cows.Therefore,we think buffalo and yellow cattle diets and living environment affect intestinal microorganisms to a certain extent.By comparing the 16S rDNA gene sequences of different species,we found the cattle specific gene markers,designed the corresponding primers and used the combination of quantitative PCR and ordinary PCR to verify the markers.The two specific primers(CFY-1 and CFY-2)could only amplify the target fragment in cow fecal.The size of the target fragment amplified by CFY-1 was 176bp and the size of the target fragment amplified by CFY-2(209bp).They have no cross reaction to the faeces of other species,and the specificity is 100%.The positive rate of CFY-2 was 93.75%(45/48)slightly higher than that of CFY-1 89.58%(43/48)and the detection limit of CFY-1(4.36×10~9copies/100mL)was slightly lower than CFY-2(6.23×10~8copies/100mL),so the sensitivity of CFY-2 is higher.In addition,we use practical cow fecal to contaminate water samples to test the practicability of CFY-1 and CFY-2,and found that both of them can effectively trace the pollution sources,CFY-2 was more effective than CFY-1(more fecal pollution sources were detected).in conclusion,we identified CFY-2as the best cow fecal gene marker.In order to explore the stability of CFY-2 specific gene markers in water environment,we used laboratory simulation cow fecal to pollute the water to explore the temperature(10°C and 30°C),water(distilled water and river water),light(sunshine24h;sunshine 14h;sunshine 10h;dark 24h)on the stability of the marker.The results showed that the time when the genetic markers were detected in distilled water at 10°C and 30°C were 80h and 48h,and then in the river at 10°C and 30°C the time were 66h and 30h.Gene markers in the sunshine 24h,sunshine 14h dark 10h,sunshine 10h dark14h,dark 24h conditions,can be detected respectively for 31h,37h,42h,54h.This study provides a highly efficient and accurate detection method for water pollution caused by cow fecal.The results of this study confirm that the detection of pollution sources and pollution levels using Faecalibacterium 16S rDNA gene marker can detect the water environment separately.It can also be used as an auxiliary detection technology to supplement the defects of traditional fecal contamination indicator so as to make the test results more persuasive.