Detection of Mycoplasma pneumoniae in respiratory samples by real-time PCR using an inhibition control

Ursi, D.; Dirven, Kristien; Loens, Katherine; Ieven, Margareta; Goossens, Herman

doi:10.1016/s0167-7012(03)00131-3

Cited by 46 publications

(45 citation statements)

References 9 publications

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“…For example, Abele-Horn et al (1998) reported that the detection limit of their assay was 3000 genome copies, 30 pg of DNA, 19 c.f.u. or 1900 organisms, and Ursi et al (2003), who used similar a method similar to ours for the real-time PCR assay, noted a detection limit of 2 c.c.u. M. pneumoniae per reaction.…”

Section: Resultsmentioning

confidence: 70%

“…Real-time PCR was performed in a Lightcycler using a Faststart DNA master hybridization probes kit (Roche Diagnostics), by a method described elsewhere (Ursi et al, 2003), except that plasmid DNA (pCRP1) containing the P1 gene of M. pneumoniae was used for the standard curve. The primers for amplification were 59-CACC CTCGGGGGCAGTCAG-39 (forward) and 59-CGGGATTCCCCG-CGGAGG-39 (reverse).…”

Section: Methodsmentioning

confidence: 99%

“…More rapid, higher sensitivity methods were therefore developed, one of them being the PCR for fragments of the P1 gene or the 16S rRNA gene (Dorigo-Zetsma et al, 1999;Tjhie et al, 1994;Ieven et al, 1996). In the last few years, real-time PCR methods for the diagnosis of M. pneumoniae have been described (Hardegger et al, 2000;Ursi et al, 2003;Templeton et al, 2003). The advantages of this application, compared to conventional PCR methods, are higher speed and less handling of PCR products such as electrophoretic analysis.…”

Section: Introductionmentioning

confidence: 99%

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Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae

Saito

Misawa

Moriya

et al. 2005

Journal of Medical Microbiology

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A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Mycoplasma pneumoniae was developed and evaluated. The assay specifically amplified only M. pneumoniae sequences, and no cross-reactivity was observed for other Mycoplasma species or respiratory bacterial species. The detection limit for this assay was found to be 2 3 10 2 copies, corresponding to 2-20 colour changing units of M. pneumoniae in 1 h, as observed in a real-time turbidimeter and electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis as well as direct sequencing of the amplified product. The assay was applied to 95 nasopharyngeal swab samples collected from patients or from healthy individuals, and compared to a real-time PCR assay in-house. A concordance of 100 % was observed between the two assays. The LAMP assay is easy to perform, shows a rapid reaction and is inexpensive. It may therefore be applied in the routine diagnosis of M. pneumoniae infection in the clinical laboratory.

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Section: Resultsmentioning

confidence: 70%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae

Saito

Misawa

Moriya

et al. 2005

Journal of Medical Microbiology

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“…Additionally, we used these RT-PCR assays to control assays for other viral pathogens, e.g., norovirus genogroups I and II, hepatitis A virus, and human immunodeficiency virus (data not shown). The incorporation of an internal control into each RT-PCR tube is important to identify inhibitors and to eliminate falsenegative results, even when problematic specimens such as stool or bronchial lavage fluids are used (9,10,22). Commercially produced diagnostic kits are available for relatively few pathogens.…”

Section: Discussionmentioning

confidence: 99%

Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays

2005

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Diagnostic systems based on reverse transcription (RT)-PCR are widely used for the detection of viral genomes in different human specimens. The application of internal controls (IC) to monitor each step of nucleic acid amplification is necessary to prevent false-negative results due to inhibition or human error. In this study, we designed various real-time RT-PCRs utilizing the coliphage MS2 replicase gene, which differ in detection format, amplicon size, and efficiency of amplification. These noncompetitive IC assays, using TaqMan, hybridization probe, or duplex scorpion probe techniques, were tested on the LightCycler and Rotorgene systems. In our approach, clinical specimens were spiked with the control virus to monitor the efficiency of extraction, reverse transcription, and amplification steps. The MS2 RT-PCR assays were applied for internal control when using a second target hepatitis C virus RNA in duplex PCR in blood donor screening. The 95% detection limit was calculated by probit analysis to 44.9 copies per PCR (range, 38.4 to 73.4). As demonstrated routinely, application of MS2 IC assays exhibits low variability and can be applied in various RT-PCR assays. MS2 phage lysates were obtained under standard laboratory conditions. The quantification of phage and template RNA was performed by plating assays to determine PFU or via real-time RT-PCR. High stability of the MS2 phage preparations stored at ؊20°C, 4°C, and room temperature was demonstrated.

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“…Real-time PCR especially is characterized by rapidity, practicability, reduced risk of contamination, and high specificity and sensitivity of detection (3,7,9,13,15,19,23,24,25). However, the increasing number of real-time PCR approaches stresses the need for comparative validation of the performance of the different test procedures.…”

mentioning

confidence: 99%

Comparison of Commercial and In-House Real-Time PCR Assays Used for Detection of Mycoplasma pneumoniae

Dumke

Jacobs

2009

J Clin Microbiol

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We tested two commercial and three in-house PCR assays under standardized conditions to detect Mycoplasma pneumoniae. All five procedures were able to demonstrate M. pneumoniae DNA in a concentration comparable to 1 CFU/l, but the mean crossing points resulted in differences in the concentration of the genome copies of a factor of 20.Recent studies have shown that the cell wall-less mollicute species Mycoplasma pneumoniae is the causative agent of about 5 to 20% of all cases of community-acquired pneumonia in humans (2). In closed populations like those in army bases or in periods showing epidemic peaks of M. pneumoniae infections (occurring every 3 to 7 years) (8, 14), the incidence of infections might increase up to 50% (2). The primarily respiratory tract infection can be followed by extrapulmonary complications (22). The specific antibiotic treatment of the disease, restricted to macrolides, tetracyclines, or fluoroquinolones, accounts for the search for sensitive, specific, and fast methods for the detection of M. pneumoniae infections in routine bacteriological practice. Cultivation is time consuming (up to 3 weeks), insensitive, and requires confirmation that the colonies grown are M. pneumoniae bacteria. Serological methods, such as the complement fixation test, enzyme-linked immunosorbent assay, and Western blot, are commonly used in the clinical laboratory but have practical limitations. The age-and timedependent formation of specific immunoglobulin A and immunoglobulin M antibodies, the persistence of detectable antibody levels resulting from previous contacts with M. pneumoniae, and the variable specificity and sensitivity of the test kits used influence the significance of the results of serodiagnosis (2). Therefore, PCR approaches extended the spectrum of available routine methods. Recent results reported the superiority of PCR over serology for the confirmation of a M. pneumoniae infection in the clinically important first week after the onset of pneumonia symptoms (17).In recent years, different PCR systems targeting a wide range of genes of M. pneumoniae (e.g., P1 adhesin, 16S rRNA, and ATPase operon gene) have been developed (16). Real-time PCR especially is characterized by rapidity, practicability, reduced risk of contamination, and high specificity and sensitivity of detection (3,7,9,13,15,19,23,24,25). However, the increasing number of real-time PCR approaches stresses the need for comparative validation of the performance of the different test procedures. Up to now, studies dealing with the evaluation of more than a single quantitative PCR system to detect M. pneumoniae have been very rare (7, 25) and have not included commercial test kits. To our knowledge, commercial quantitative PCR systems for the detection of M. pneumoniae are not available in the United States (2), whereas kits from different manufacturers are widely used in Europe. The aim of the present study was to investigate the sensitivities of different commercial and in-house real-time PCR approaches for the detection of M. p...

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Detection of Mycoplasma pneumoniae in respiratory samples by real-time PCR using an inhibition control

Cited by 46 publications

References 9 publications

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae

Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays

Comparison of Commercial and In-House Real-Time PCR Assays Used for Detection of Mycoplasma pneumoniae

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