title: Employment of Near Full-Length Ribosome Gene TA-Cloning and Primer-Blast to Detect Multiple Species in a Natural Complex Microbial Community Using Species-Specific Primers Designed with Their Genome Sequences.
abstract: It remains an unsolved problem to quantify a natural microbial community by rapidly and conveniently measuring multiple species with functional significance. Most widely used high throughput next-generation sequencing methods can only generate information mainly for genus-level taxonomic identification and quantification, and detection of multiple species in a complex microbial community is still heavily dependent on approaches based on near full-length ribosome RNA gene or genome sequence information. In this study, we used near full-length rRNA gene library sequencing plus Primer-Blast to design species-specific primers based on whole microbial genome sequences. The primers were intended to be specific at the species level within relevant microbial communities, i.e., a defined genomics background. The primers were tested with samples collected from the Daqu (also called fermentation starters) and pit mud of a traditional Chinese liquor production plant. Sixteen pairs of primers were found to be suitable for identification of individual species. Among them, seven pairs were chosen to measure the abundance of microbial species through quantitative PCR. The combination of near full-length ribosome RNA gene library sequencing and Primer-Blast may represent a broadly useful protocol to quantify multiple species in complex microbial population samples with species-specific primers.
authors.affiliation: School of Chemical Engineering & Technology, Marine Antifouling Engineering Technology Center of Shandong Province, Harbin Institute of Technology, Harbin, 150006, China.|Department of Biological Sciences, Bowling Green State University, Bowling Green, OH, 43403-0208, USA.
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhang
authors.affiliation: The GuJing Group, Bozhou, 236800, Anhui, China.
authors.forename: Hongkui
authors.initials: H
authors.lastname: He
authors.affiliation: The GuJing Group, Bozhou, 236800, Anhui, China.
authors.forename: Xiujuan
authors.initials: X
authors.lastname: Yu
authors.affiliation: Department of Biological Sciences, Bowling Green State University, Bowling Green, OH, 43403-0208, USA. zxu@bgsu.edu.
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Xu
authors.affiliation: School of Chemical Engineering & Technology, Marine Antifouling Engineering Technology Center of Shandong Province, Harbin Institute of Technology, Harbin, 150006, China. zhangzzbiox@hitwh.edu.cn.|Shandong Gredmedic Co., Ltd., Weihai, China. zhangzzbiox@hitwh.edu.cn.
authors.forename: Zhizhou
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D017931:DNA Primers / Q000235:genetics*; D005516:Food Microbiology; D015723:Gene Library; D054892:Metagenome; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D012329:RNA, Bacterial; D012335:RNA, Ribosomal
keywords: Full-length rRNA; Genome sequence; Primer-Blast; Species-specific primer; qPCR