title: PCR-Based Multiple Species Cell Counting for In Vitro Mixed Culture.
abstract: Changes of bacterial profiles in microbial communities are strongly associated with human health. There is an increasing need for multiple species research in vitro. To avoid high cost or measurement of a limited number of species, PCR-based multiple species cell counting (PCR-MSCC) has been conceived. Species-specific sequence is defined as a unique sequence of one species in a multiple species mixed culture. This sequence is identified by comparing a random 1000 bp genomic sequence of one species with the whole genome sequences of the other species in the same artificial mixed culture. If absent in the other genomes, it is the species-specific sequence. Species-specific primers were designed based on the species-specific sequences. In the present study, ten different oral bacterial species were mixed and grown in Brain Heart Infusion Yeast Extract with 1% sucrose for 24 hours. Biofilm was harvested and processed for DNA extraction and q-PCR amplification with the species-specific primers. By comparing the q-PCR data of each species in the unknown culture with reference cultures, in which the cell number of each species was determined by colony forming units on agar plate, the cell number of that strain in the unknown mixed culture was calculated. This technique is reliable to count microorganism numbers that are less than 100,000 fold different from other species within the same culture. Theoretically, it can be used in detecting a species in a mixed culture of over 200 species. Currently PCR-MSCC is one of the most economic methods for quantifying single species cell numbers, especially for the low abundant species, in a multiple artificial mixed culture in vitro.
authors.affiliation: Department of Pediatric Dentistry, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China; Department of Oral Biology, School of Dentistry, Indiana University, Indianapolis, Indiana, United States of America; State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China.
authors.forename: Ruijie
authors.initials: R
authors.lastname: Huang
authors.affiliation: Department of Microbiology and Immunology, School of Medicine, Indiana University, Indianapolis, Indiana, United States of America.
authors.forename: Junjie
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Microbiology and Immunology, School of Medicine, Indiana University, Indianapolis, Indiana, United States of America.
authors.forename: X Frank
authors.initials: XF
authors.lastname: Yang
authors.affiliation: Department of Oral Biology, School of Dentistry, Indiana University, Indianapolis, Indiana, United States of America; Department of Pathology and Laboratory Medicine, School of Medicine, Indiana University, Indianapolis, Indiana, United States of America.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Gregory
mesh_terms: D001483:Base Sequence; D046508:Culture Techniques; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial; D064307:Microbiota / Q000235:genetics*; D058889:Molecular Typing / Q000379:methods*; D009055:Mouth / Q000382:microbiology; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013291:Streptococcus / Q000235:genetics
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial