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New Diagnostic Real-Time PCR for Specific Detection of Mycoplasma hominis DNA.

Pascual A, Jaton K, Ninet B, Bille J, Greub G - Int J Microbiol (2010)

Bottom Line: Mycoplasma hominis is a fastidious micro-organism causing genital and extragenital infections.We developed a specific real-time PCR that exhibits high sensitivity and low intrarun and interrun variabilities.When applied to clinical samples, this quantitative PCR allowed to confirm the role of M. hominis in three patients with severe extragenital infections.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases Service, Department of Internal Medicine, University Hospital Center and University of Lausanne, 1011 Lausanne, Switzerland.

ABSTRACT
Mycoplasma hominis is a fastidious micro-organism causing genital and extragenital infections. We developed a specific real-time PCR that exhibits high sensitivity and low intrarun and interrun variabilities. When applied to clinical samples, this quantitative PCR allowed to confirm the role of M. hominis in three patients with severe extragenital infections.

No MeSH data available.


Related in: MedlinePlus

Intra and  interrun reproducibility of the real-time PCR assessed on duplicate of plasmid positive controls performed at 10-fold dilutions from 1000 to 10 plasmid copies/5 μL during 10 successive runs. (a) Plots of the cycle threshold (Ct) of first and second duplicates, showing intrarun variability of the real-time PCR between duplicates of positive plasmid controls; 95% confidence interval is shown by the dashed lines. (b) Bland-Altman graph showing the ratio of Ct of both duplicates according to the mean of the Ct of duplicates. (c) Plots of the mean of duplicate of plasmid positive controls according to each successive run, showing the low interrun variability of the real-time PCR. Standard deviations show the intrarun reproducibility of the PCR.
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fig1: Intra and interrun reproducibility of the real-time PCR assessed on duplicate of plasmid positive controls performed at 10-fold dilutions from 1000 to 10 plasmid copies/5 μL during 10 successive runs. (a) Plots of the cycle threshold (Ct) of first and second duplicates, showing intrarun variability of the real-time PCR between duplicates of positive plasmid controls; 95% confidence interval is shown by the dashed lines. (b) Bland-Altman graph showing the ratio of Ct of both duplicates according to the mean of the Ct of duplicates. (c) Plots of the mean of duplicate of plasmid positive controls according to each successive run, showing the low interrun variability of the real-time PCR. Standard deviations show the intrarun reproducibility of the PCR.

Mentions: Duplicates of 10-fold serial dilutions of the plasmid were run in ten independent experiments in order to analyze the sensitivity and the reproducibility of the PCR results. As shown in Figure 1(a), the intrarun reproducibility was excellent. As expected, intrarun variability was slightly higher at very low concentration of target DNA (Figure 1(b)). The interrun variability was low, except at a low concentration of 10 plasmid copies per 5 μl (Figure 1(c)). Thus, the mean Ct values +/−1 standard deviation were of 29.64 +/− 0.38 (1.28%), 32.69 +/− 0.36 (1.10%), and 36.43 +/− 0.49 (1.34%) for 1000, 100, and 10 DNA copies per 5 μl. The analytical sensitivity of the real-time PCR was 10 copies of plasmid control DNA per reaction mixture, that is, similar to the light-cycler PCR already reported by Baczynska et al. [14]. This sensitivity is about 10-fold better than the 16SrRNA broad-range PCR developed by Bosshard et al. [15]. When testing genomic DNA obtained by growing M. hominis in culture, we again obtained an excellent sensitivity which was of about 1000 bacteria/ml. When serially diluting a clinical sample (inguinal abscess of patient 1), the eubacterial PCR was slightly positive when the real-time specific Mycoplasma PCR was positive with a Ct of 33.4 (45 copies in 5 μl of DNA) whereas eubacterial PCR was negative when the real-time specific Mycoplasma PCR was positive with a Ct of 36.3 (6.5 copies in 5 μl of DNA).


New Diagnostic Real-Time PCR for Specific Detection of Mycoplasma hominis DNA.

Pascual A, Jaton K, Ninet B, Bille J, Greub G - Int J Microbiol (2010)

Intra and  interrun reproducibility of the real-time PCR assessed on duplicate of plasmid positive controls performed at 10-fold dilutions from 1000 to 10 plasmid copies/5 μL during 10 successive runs. (a) Plots of the cycle threshold (Ct) of first and second duplicates, showing intrarun variability of the real-time PCR between duplicates of positive plasmid controls; 95% confidence interval is shown by the dashed lines. (b) Bland-Altman graph showing the ratio of Ct of both duplicates according to the mean of the Ct of duplicates. (c) Plots of the mean of duplicate of plasmid positive controls according to each successive run, showing the low interrun variability of the real-time PCR. Standard deviations show the intrarun reproducibility of the PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2913506&req=5

fig1: Intra and interrun reproducibility of the real-time PCR assessed on duplicate of plasmid positive controls performed at 10-fold dilutions from 1000 to 10 plasmid copies/5 μL during 10 successive runs. (a) Plots of the cycle threshold (Ct) of first and second duplicates, showing intrarun variability of the real-time PCR between duplicates of positive plasmid controls; 95% confidence interval is shown by the dashed lines. (b) Bland-Altman graph showing the ratio of Ct of both duplicates according to the mean of the Ct of duplicates. (c) Plots of the mean of duplicate of plasmid positive controls according to each successive run, showing the low interrun variability of the real-time PCR. Standard deviations show the intrarun reproducibility of the PCR.
Mentions: Duplicates of 10-fold serial dilutions of the plasmid were run in ten independent experiments in order to analyze the sensitivity and the reproducibility of the PCR results. As shown in Figure 1(a), the intrarun reproducibility was excellent. As expected, intrarun variability was slightly higher at very low concentration of target DNA (Figure 1(b)). The interrun variability was low, except at a low concentration of 10 plasmid copies per 5 μl (Figure 1(c)). Thus, the mean Ct values +/−1 standard deviation were of 29.64 +/− 0.38 (1.28%), 32.69 +/− 0.36 (1.10%), and 36.43 +/− 0.49 (1.34%) for 1000, 100, and 10 DNA copies per 5 μl. The analytical sensitivity of the real-time PCR was 10 copies of plasmid control DNA per reaction mixture, that is, similar to the light-cycler PCR already reported by Baczynska et al. [14]. This sensitivity is about 10-fold better than the 16SrRNA broad-range PCR developed by Bosshard et al. [15]. When testing genomic DNA obtained by growing M. hominis in culture, we again obtained an excellent sensitivity which was of about 1000 bacteria/ml. When serially diluting a clinical sample (inguinal abscess of patient 1), the eubacterial PCR was slightly positive when the real-time specific Mycoplasma PCR was positive with a Ct of 33.4 (45 copies in 5 μl of DNA) whereas eubacterial PCR was negative when the real-time specific Mycoplasma PCR was positive with a Ct of 36.3 (6.5 copies in 5 μl of DNA).

Bottom Line: Mycoplasma hominis is a fastidious micro-organism causing genital and extragenital infections.We developed a specific real-time PCR that exhibits high sensitivity and low intrarun and interrun variabilities.When applied to clinical samples, this quantitative PCR allowed to confirm the role of M. hominis in three patients with severe extragenital infections.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases Service, Department of Internal Medicine, University Hospital Center and University of Lausanne, 1011 Lausanne, Switzerland.

ABSTRACT
Mycoplasma hominis is a fastidious micro-organism causing genital and extragenital infections. We developed a specific real-time PCR that exhibits high sensitivity and low intrarun and interrun variabilities. When applied to clinical samples, this quantitative PCR allowed to confirm the role of M. hominis in three patients with severe extragenital infections.

No MeSH data available.


Related in: MedlinePlus