title: Evaluation of Faecalibacterium 16S rDNA genetic markers for accurate identification of swine faecal waste by quantitative PCR.
abstract: A genetic marker within the 16S rRNA gene of Faecalibacterium was identified for use in a quantitative PCR (qPCR) assay to detect swine faecal contamination in water. A total of 146,038 bacterial sequences were obtained using 454 pyrosequencing. By comparative bioinformatics analysis of Faecalibacterium sequences with those of numerous swine and other animal species, swine-specific Faecalibacterium 16S rRNA gene sequences were identified and Polymerase Chain Okabe (PCR) primer sets designed and tested against faecal DNA samples from swine and non-swine sources. Two PCR primer sets, PFB-1 and PFB-2, showed the highest specificity to swine faecal waste and had no cross-reaction with other animal samples. PFB-1 and PFB-2 amplified 16S rRNA gene sequences from 50 samples of swine with positive ratios of 86 and 90%, respectively. We compared swine-specific Faecalibacterium qPCR assays for the purpose of quantifying the newly identified markers. The quantification limits (LOQs) of PFB-1 and PFB-2 markers in environmental water were 6.5 and 2.9 copies per 100 ml, respectively. Of the swine-associated assays tested, PFB-2 was more sensitive in detecting the swine faecal waste and quantifying the microbial load. Furthermore, the microbial abundance and diversity of the microbiomes of swine and other animal faeces were estimated using operational taxonomic units (OTUs). The species specificity was demonstrated for the microbial populations present in various animal faeces.
authors.affiliation: Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, PR China. Electronic address: chrduan@cqu.edu.cn.
authors.forename: Chuanren
authors.initials: C
authors.lastname: Duan
authors.affiliation: Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, PR China.
authors.forename: Yamin
authors.initials: Y
authors.lastname: Cui
authors.affiliation: Key Laboratory of Drug Targeting and Drug Delivery System of Education Ministry, Department of Medicinal Chemistry, West China School of Pharmacy, Sichuan University, Chengdu, 610041, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Key Laboratory of the Three Gorges Reservoir Region's Eco-Environment, College of Urban Construction and Environmental Engineering, Chongqing University, Chongqing, 400045, PR China. Electronic address: zhaijun99@126.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhai
authors.affiliation: Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, PR China.
authors.forename: Baoyun
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, PR China.
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, PR China.
authors.forename: Da
authors.initials: D
authors.lastname: Sun
authors.affiliation: Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, PR China.
authors.forename: Hang
authors.initials: H
authors.lastname: Chen
mesh_terms: D000818:Animals; D002417:Cattle; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal; D004285:Dogs; D000070036:Faecalibacterium / Q000235:genetics*; D005243:Feces / Q000382:microbiology*; D005819:Genetic Markers; D006801:Humans; D064307:Microbiota / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013552:Swine / Q000382:microbiology*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D005819:Genetic Markers; D012336:RNA, Ribosomal, 16S
keywords: 454 pyrosequencing; Faecalibacterium; Quantitative PCR; Swine faecal waste