PCR-based multiple species cell counting for in vitro mixed culture

Ruijie Huang, Junjie Zhang, X. Yang, Richard Gregory

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere0126628
JournalPLoS One
Volume10
Issue number5
DOIs
StatePublished - May 13 2015

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mixed culture
Genes
Polymerase Chain Reaction
Cell Count
Biofilms
Cell culture
Microorganisms
Yeast
Agar
Amplification
Sucrose
Brain
Health
Genome
cells
Economics
DNA
genome
Costs
yeast extract

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

PCR-based multiple species cell counting for in vitro mixed culture. / Huang, Ruijie; Zhang, Junjie; Yang, X.; Gregory, Richard.

In: PLoS One, Vol. 10, No. 5, e0126628, 13.05.2015.

Research output: Contribution to journalArticle

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