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Direct Detection and Quantification of Bacterial Genes Associated with Inflammation in DNA Isolated from Stool.

Gómez-Moreno R, Robledo IE, Baerga-Ortiz A - Adv Microbiol (2014)

Bottom Line: Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation.Our results show that 20% of the samples (N = 41) tested positive for detectable amounts of pks island genes, whereas 10% of individuals were positive for tcpC or gelE and only one individual was found to harbor the cnf-1 gene.Of the 13 individuals that were positive for at least one of the pro-inflammatory genes, 5 were found to harbor more than one.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico ; Molecular Sciences Building, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico.

ABSTRACT

Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation. In this work, we report a method for the direct detection and quantification of these pro-inflammatory bacterial genes by PCR and qPCR in DNA extracted from human stool samples. PCR reactions were performed to detect (i) the pks island genes, (ii) tcpC, which is present in some strains of Escherichia coli and (iii) gelE presented in some strains of Enterococcus faecalis. Additionally, we screened for the presence of the following genes encoding cyclomodulins that disrupted mammalian cell division: (iv) cdt (which encodes the cytolethal distending toxin) and (v) cnf-1 (which encodes the cytotoxic necrotizing factor-1). Our results show that 20% of the samples (N = 41) tested positive for detectable amounts of pks island genes, whereas 10% of individuals were positive for tcpC or gelE and only one individual was found to harbor the cnf-1 gene. Of the 13 individuals that were positive for at least one of the pro-inflammatory genes, 5 were found to harbor more than one. A quantitative version of the assay, which used real-time PCR, revealed the pro-inflammatory genes to be in high copy numbers: up to 1.3 million copies per mg of feces for the pks island genes. Direct detection of specific genes in stool could prove useful toward screening for the presence of pro-inflammatory bacterial genes in individuals with inflammatory bowel diseases or colorectal cancer.

No MeSH data available.


Related in: MedlinePlus

For the quantification of DNA copy number, a standard curve was generated for each of the pro-inflammatory genes as described in the methods section. Standards containing 393 ng/μl (77,058,825 copies per μl), 100 ng/μl (19,607,843 copies per μl), 10 ng/μl (1,960,784 copies per μl), 1 ng/μl (196,078 copies per μl) and 0.1 ng/μl (19,607 copies per μl) were used to make the calibration curve. Typical copy number estimates for tcpC-positive samples ranged from 100,000 – 800,000 copies per μl. In this figure we show (a) the agarose gel electrophoresis for the PCR amplification of tcpC gene using different known amounts of E. coli DNA in the reaction mixture, (b) the qPCR reaction for the standards monitored in real time and (c) the calibration curve for the quantification of tcpC copies.
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Figure 3: For the quantification of DNA copy number, a standard curve was generated for each of the pro-inflammatory genes as described in the methods section. Standards containing 393 ng/μl (77,058,825 copies per μl), 100 ng/μl (19,607,843 copies per μl), 10 ng/μl (1,960,784 copies per μl), 1 ng/μl (196,078 copies per μl) and 0.1 ng/μl (19,607 copies per μl) were used to make the calibration curve. Typical copy number estimates for tcpC-positive samples ranged from 100,000 – 800,000 copies per μl. In this figure we show (a) the agarose gel electrophoresis for the PCR amplification of tcpC gene using different known amounts of E. coli DNA in the reaction mixture, (b) the qPCR reaction for the standards monitored in real time and (c) the calibration curve for the quantification of tcpC copies.

Mentions: From the PCR results visualized on an agarose gel, it was clear that some samples registered a more intense band than others, raising the possibility that some individuals harbor a higher content of bacterial pro-inflammatory genes than others. In order to provide a quantitative estimate of the number of copies of the pro-inflammatory genes per patient, we performed the PCR assay with real-time detection (Figure 3). A standard curve that was made with known amounts of DNA copies as described in the methods section, enabled the quantification of DNA copy number for each of the genes in the 41 stool samples. The number of copies of pks island among those who tested positive fluctuated between 2800 and 1.3 million copies per mg of stool analyzed and the number of gelE copies fluctuated between 6000 – 260,000 copies per mg of stool. The amount of tcpC DNA was less variable fluctuating between 100,000 – 800,000 copies per mg of stool. Future work will be aimed at establishing whether a correlation exists between a high copy number for these pro-inflammatory genes and a higher risk of developing gastrointestinal disorders.


Direct Detection and Quantification of Bacterial Genes Associated with Inflammation in DNA Isolated from Stool.

Gómez-Moreno R, Robledo IE, Baerga-Ortiz A - Adv Microbiol (2014)

For the quantification of DNA copy number, a standard curve was generated for each of the pro-inflammatory genes as described in the methods section. Standards containing 393 ng/μl (77,058,825 copies per μl), 100 ng/μl (19,607,843 copies per μl), 10 ng/μl (1,960,784 copies per μl), 1 ng/μl (196,078 copies per μl) and 0.1 ng/μl (19,607 copies per μl) were used to make the calibration curve. Typical copy number estimates for tcpC-positive samples ranged from 100,000 – 800,000 copies per μl. In this figure we show (a) the agarose gel electrophoresis for the PCR amplification of tcpC gene using different known amounts of E. coli DNA in the reaction mixture, (b) the qPCR reaction for the standards monitored in real time and (c) the calibration curve for the quantification of tcpC copies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307837&req=5

Figure 3: For the quantification of DNA copy number, a standard curve was generated for each of the pro-inflammatory genes as described in the methods section. Standards containing 393 ng/μl (77,058,825 copies per μl), 100 ng/μl (19,607,843 copies per μl), 10 ng/μl (1,960,784 copies per μl), 1 ng/μl (196,078 copies per μl) and 0.1 ng/μl (19,607 copies per μl) were used to make the calibration curve. Typical copy number estimates for tcpC-positive samples ranged from 100,000 – 800,000 copies per μl. In this figure we show (a) the agarose gel electrophoresis for the PCR amplification of tcpC gene using different known amounts of E. coli DNA in the reaction mixture, (b) the qPCR reaction for the standards monitored in real time and (c) the calibration curve for the quantification of tcpC copies.
Mentions: From the PCR results visualized on an agarose gel, it was clear that some samples registered a more intense band than others, raising the possibility that some individuals harbor a higher content of bacterial pro-inflammatory genes than others. In order to provide a quantitative estimate of the number of copies of the pro-inflammatory genes per patient, we performed the PCR assay with real-time detection (Figure 3). A standard curve that was made with known amounts of DNA copies as described in the methods section, enabled the quantification of DNA copy number for each of the genes in the 41 stool samples. The number of copies of pks island among those who tested positive fluctuated between 2800 and 1.3 million copies per mg of stool analyzed and the number of gelE copies fluctuated between 6000 – 260,000 copies per mg of stool. The amount of tcpC DNA was less variable fluctuating between 100,000 – 800,000 copies per mg of stool. Future work will be aimed at establishing whether a correlation exists between a high copy number for these pro-inflammatory genes and a higher risk of developing gastrointestinal disorders.

Bottom Line: Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation.Our results show that 20% of the samples (N = 41) tested positive for detectable amounts of pks island genes, whereas 10% of individuals were positive for tcpC or gelE and only one individual was found to harbor the cnf-1 gene.Of the 13 individuals that were positive for at least one of the pro-inflammatory genes, 5 were found to harbor more than one.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico ; Molecular Sciences Building, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico.

ABSTRACT

Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation. In this work, we report a method for the direct detection and quantification of these pro-inflammatory bacterial genes by PCR and qPCR in DNA extracted from human stool samples. PCR reactions were performed to detect (i) the pks island genes, (ii) tcpC, which is present in some strains of Escherichia coli and (iii) gelE presented in some strains of Enterococcus faecalis. Additionally, we screened for the presence of the following genes encoding cyclomodulins that disrupted mammalian cell division: (iv) cdt (which encodes the cytolethal distending toxin) and (v) cnf-1 (which encodes the cytotoxic necrotizing factor-1). Our results show that 20% of the samples (N = 41) tested positive for detectable amounts of pks island genes, whereas 10% of individuals were positive for tcpC or gelE and only one individual was found to harbor the cnf-1 gene. Of the 13 individuals that were positive for at least one of the pro-inflammatory genes, 5 were found to harbor more than one. A quantitative version of the assay, which used real-time PCR, revealed the pro-inflammatory genes to be in high copy numbers: up to 1.3 million copies per mg of feces for the pks island genes. Direct detection of specific genes in stool could prove useful toward screening for the presence of pro-inflammatory bacterial genes in individuals with inflammatory bowel diseases or colorectal cancer.

No MeSH data available.


Related in: MedlinePlus