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PCR-Based Multiple Species Cell Counting for In Vitro Mixed Culture.

Huang R, Zhang J, Yang XF, Gregory RL - PLoS ONE (2015)

Bottom Line: Changes of bacterial profiles in microbial communities are strongly associated with human health.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

q-PCR efficiency test.The x-axis represents 1:2n dilutions of DNA and the y-axis represents Ct values. Ideally, if one sample is diluted 1:2 (Δx = 1), it will take one more cycle (Δy = 1) to reach the same threshold. Ideally, the slope (Δy / Δx), which represents the amplification efficiency E, is equal to 1. For actual samples the slope is less than 1.
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pone.0126628.g003: q-PCR efficiency test.The x-axis represents 1:2n dilutions of DNA and the y-axis represents Ct values. Ideally, if one sample is diluted 1:2 (Δx = 1), it will take one more cycle (Δy = 1) to reach the same threshold. Ideally, the slope (Δy / Δx), which represents the amplification efficiency E, is equal to 1. For actual samples the slope is less than 1.

Mentions: The q-PCR efficiency test implied the median E is 82.7% with the range from 70.0% to 98.5% (Table 3). The coefficient of determination (R2) of every regression line was equal to or larger than 0.95 (Fig 3). For most of the q-PCR efficiency studies, DNA template concentrations (log10X) were used as the X-axis, and the efficiency calculation equation was E = 10−1/slope−1 with the ideal slope of -3.32 [17–19]. In the present study, we changed the X-axis to 1:2n dilutions of DNA template, this made the efficiency much easier to predict because E = slope. However, if the absolute DNA template will be used, the X-axis could be stated as (log2X)−1. R2 in the present study could approach 1 by adding five more DNA template dilution points for the linear regression [18,19]. The amplification condition of each species was not optimized because in optimizing there would be 10 separate PCR amplifications, one for each species. It is practical with 10 species, but may not be practical with more than 20 species. In a long run point of view, the default PCR set-up instead of an optimized condition for each species was used.


PCR-Based Multiple Species Cell Counting for In Vitro Mixed Culture.

Huang R, Zhang J, Yang XF, Gregory RL - PLoS ONE (2015)

q-PCR efficiency test.The x-axis represents 1:2n dilutions of DNA and the y-axis represents Ct values. Ideally, if one sample is diluted 1:2 (Δx = 1), it will take one more cycle (Δy = 1) to reach the same threshold. Ideally, the slope (Δy / Δx), which represents the amplification efficiency E, is equal to 1. For actual samples the slope is less than 1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430427&req=5

pone.0126628.g003: q-PCR efficiency test.The x-axis represents 1:2n dilutions of DNA and the y-axis represents Ct values. Ideally, if one sample is diluted 1:2 (Δx = 1), it will take one more cycle (Δy = 1) to reach the same threshold. Ideally, the slope (Δy / Δx), which represents the amplification efficiency E, is equal to 1. For actual samples the slope is less than 1.
Mentions: The q-PCR efficiency test implied the median E is 82.7% with the range from 70.0% to 98.5% (Table 3). The coefficient of determination (R2) of every regression line was equal to or larger than 0.95 (Fig 3). For most of the q-PCR efficiency studies, DNA template concentrations (log10X) were used as the X-axis, and the efficiency calculation equation was E = 10−1/slope−1 with the ideal slope of -3.32 [17–19]. In the present study, we changed the X-axis to 1:2n dilutions of DNA template, this made the efficiency much easier to predict because E = slope. However, if the absolute DNA template will be used, the X-axis could be stated as (log2X)−1. R2 in the present study could approach 1 by adding five more DNA template dilution points for the linear regression [18,19]. The amplification condition of each species was not optimized because in optimizing there would be 10 separate PCR amplifications, one for each species. It is practical with 10 species, but may not be practical with more than 20 species. In a long run point of view, the default PCR set-up instead of an optimized condition for each species was used.

Bottom Line: Changes of bacterial profiles in microbial communities are strongly associated with human health.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus