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.

Dissecting individual pathogen-commensal interactions within a complex gut microbiota community

“…Primers were designed to target either the topoisomerase I ( topI ) or DNA gyrase subunit A ( gyrA ) region of each strain (Supp Table 1 for sequences). The primers were designed using Primer-BLAST, this enables a high level of species specificity to be engineered in situ 36,37 . Primers were designed to create an amplicon of 100-150 bases, with an annealing temperature of 59-60°C, all primers were screened against the ‘nt’ database for specificity.…”

“…To convert from a qPCR output cycle threshold (Ct) to a predicted bacterial number we utilized previously described methods 32,37 . This required creating a standard curve for each species, the gradient of which is related to the primer amplification efficiency.…”

Bioconversion of distillers’ grains hydrolysates to advanced biofuels by an Escherichia coli co-culture

“…The cell numbers of E. coli BLF2 and AY3 in the co-culture were determined by the PCR-based multiple species cell counting method as described by Huang et al [ 22 ]. To prepare the reference mixed samples, E. coli BLF2 and AY3 were grown overnight in 3 mL LB medium respectively.…”

“…The cell numbers of E. coli BLF2 and AY3 in the co-culture samples during the fermentation process are determined by the following equation modified from [ 22 ] (the genomic DNA of the reference samples and unknown co-culture samples have the same dilution for q-PCR reaction): where N X = cell number of E. coli BLF2 or AY3 in the co-culture; E = amplification efficiency of the q-PCR reaction using the primers specific to BLF2 or AY3; C T,R = the threshold cycles ( C T ) of q-PCR for BLF2 or AY3 in the reference sample; C T,X = the threshold cycles ( C T ) of q-PCR for BLF2 or AY3 in the unknown co-culture sample; CFU R = the cell concentration of BLF2 or AY3 reference sample; V R = the volume of processed reference cells for DNA extraction.…”

Sulfated vizantin causes detachment of biofilms composed mainly of the genus Streptococcus without affecting bacterial growth and viability

“…To determine the species composition of the genus Streptococcus in the CB and GB, q-PCR analysis was performed as described previously [46][47][48]. Briefly, standard curves of representative species were first created.…”