Activity and DNA contamination of commercial polymerase chain reaction reagents for the universal 16S rDNA real-time polymerase chain reaction detection of bacterial pathogens in blood

Mühl, Helge; Kochem, Anna-Julia; Disqué, Claudia; Sakka, Samir G.

doi:10.1016/j.diagmicrobio.2008.07.011

Cited by 85 publications

(53 citation statements)

References 26 publications

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“…were noted. Unfortunately, with 16S rRNA gene PCR, some false-positive results can also arise directly from contaminants in the molecular reagents (19)(20)(21). During our study, several problems of this type occurred; contamination of the premix by Pseudomonas orientalis affected two laboratories using the same lot of this reagent or Acinetobacter spp.…”

Section: Discussionmentioning

confidence: 91%

First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

Plouzeau

Bémer²,

Valentin

et al. 2015

J Clin Microbiol

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The objective of this study was to assess the performance of seven French laboratories for 16S rRNA gene detection by real-time PCR in the diagnosis of bone and joint infection (BJI) to validate a large multicenter study. External quality control (QC) was required owing to the differences in extraction procedures and the molecular equipment used in the different laboratories. Three proficiency sets were organized, including four bacterial DNA extracts and four bead mill-pretreated osteoarticular specimens. Extraction volumes, 16S rRNA gene primers, and sequencing interpretation rules were standardized. In order to assess each laboratory's ability to achieve the best results, scores were assigned, and each QC series was classified as optimal, acceptable, or to be improved. A total of 168 QCs were sent, and 160 responses were analyzed. The expected results were obtained for 93.8%, with the same proportion for extracts (75/80) and clinical specimens (75/80). For the specimens, there was no significant difference between manual and automated extraction. This QC demonstrated the ability to achieve good and homogeneous results using the same 16S rRNA gene PCR with different equipment and validates the possibility of high-quality multicenter studies using molecular diagnosis for BJI.

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Section: Discussionmentioning

confidence: 91%

First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

Plouzeau

Bémer²,

Valentin

et al. 2015

J Clin Microbiol

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“…Pathogen identification was done using MALDI-TOF MS (Bruker Daltonic GmbH, Germany). The diagnosis of culture-positivity was based on the IDSA recommendations regarding the management of PJI [17]. In brief, two or more culture samples with the identical pathogen were considered as definitive positive.…”

Section: Conventional Culture Microbiologymentioning

confidence: 99%

“…Inhibition of the PCR was excluded by adding internal controls to each sample extract. A sample was considered PCR positive if the melting curve analysis showed a peak within the expected Tm range [17]. Amplicons from positive PCR reactions were purified with GFX PCR DNA Purification kit.…”

Section: Conventional Culture Microbiologymentioning

confidence: 99%

Diagnosis of prosthetic joint infections using UMD-Universal Kit and the automated multiplex-PCR Unyvero i60 ITI® cartridge system: a pilot study

et al. 2015

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cultures of periprosthetic tissue samples were compared with the results of broad-range 16S rRNA gene real-time PCR (UMD-Universal Pathogen DNA Extraction and PCR Analysis, Molzym GmbH, Germany) and the multiplex-PCR Unyvero ITI ® cartridge system (U-ITI; Curetis AG, Germany). Conventional culture and broad-range 16S rRNA gene real-time PCR were performed on all samples. U-ITI was used in a subgroup of 28 cases including all culture-positive cases. The agreement of the results from the methods was assessed. Results Of 54 cases, seven were culture-positive. Broadrange 16S rRNA gene real-time PCR gave 6, U-ITI 3 concordant positive results. Of the 47 culture-negative samples, 46 were also negative by broad-range 16S rRNA gene realtime PCR resulting in a 96 % (52/54) agreement between 16S rRNA gene PCR and culture. Of the 21 culture-negative samples analysed with U-ITI, 20 gave negative results, including the single 16S rRNA gene PCR-positive/culturenegative specimen. The rate of agreement between U-ITI and culture results was 82 % (23/28). Conclusion This pilot study gave no indication of superiority of the used NAATs over conventional culture methods for the microbiological diagnosis of PJI. Drawbacks are susceptibility to contamination in the case of 16S rRNA gene real-time PCR, labour-intensive DNA extraction and limited pathogen panel in the case of the multiplex cartridge PCR system. More prospective trials are needed to evaluate the diagnostic performance of NAATs and their impact on the clinical management of PJI. Keywords

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“…The high sensitivity of detection by PCR of bacterial DNA (15) suggests its use in the diagnosis of bacteremia (16). Initial disadvantages of PCR, notably the incidence of false-positive results from bacterial DNA contaminating PCR reagents (4,39), have been counteracted by the development of purification methods (12,28) and the availability of commercial products (22).…”

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confidence: 99%

Diagnosis of Bacteremia in Whole-Blood Samples by Use of a Commercial Universal 16S rRNA Gene-Based PCR and Sequence Analysis

et al. 2009

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In a prospective, multicenter study of 342 blood samples from 187 patients with systemic inflammatory response syndrome, sepsis, or neutropenic fever, a new commercial PCR test (SepsiTest; Molzym) was evaluated for rapid diagnosis of bacteremia. The test comprises a universal PCR from the 16S rRNA gene, with subsequent identification of bacteria from positive samples by sequence analysis of amplicons. Compared to blood culture (BC), the diagnostic sensitivity and specificity of the PCR were 87.0 and 85.8%, respectively. Considering the 34 BC-positive patients, 28 were also PCR positive in at least one of the samples, resulting in a patient-related sensitivity of 82.4%. The concordance of PCR and BC for both positive and negative samples was (47 ؉ 247)/342, i.e., 86.0%. In total, 31 patients were PCR/sequencing positive and BC negative, in whom the PCR result was judged as possible or probable to true bacteremia in 25. In conclusion, the PCR approach facilitates the detection of bacteremia in blood samples within a few hours. Despite the indispensability of BC diagnostics, the rapid detection of bacteria by SepsiTest appears to be a valuable tool, allowing earlier pathogen-adapted antimicrobial therapy in critically ill patients.Bloodstream infection is a life-threatening condition with a high mortality rate, especially in intensive care and neutropenic patients (5,19,35,38). Pathogenic bacteria are the most frequent causes of bloodstream infection, although fungi can also be isolated in a minority of patients (7,17,21,32,34). Currently, inoculation of blood cultures (BC) is the standard method for microbiological diagnosis of bloodstream infections. However, the limitations of BC include relatively low sensitivities and a long time-to-result for detection and identification of the pathogen, generally over 2 days, and even longer for fastidious organisms (13,20,27).In contrast, DNA-based procedures may offer faster and more reliable diagnoses (3, 30). PCR amplification of microbial genes, followed by detection of amplified products by gel electrophoresis or real-time PCR monitoring using fluorescent dyes or target-directed fluorescent probes, is a quick process allowing pathogen detection within a few hours (18). Identification of microorganisms can be performed by PCR algorithms, taxon-specific oligonucleotide microarrays, or sequencing amplicons (30).PCR amplification of conserved regions of the bacterial genome, in particular the 16S rRNA gene, combined with sequence analysis is a well-established technique for the identification of bacterial pathogens (18). The main advantages of targeting the 16S rRNA gene are the broad range of pathogens detectable and the independence of this method from the in vitro viability of strains (6). The high sensitivity of detection by PCR of bacterial DNA (15) suggests its use in the diagnosis of bacteremia (16). Initial disadvantages of PCR, notably the incidence of false-positive results from bacterial DNA contaminating PCR reagents (4, 39), have been counteracted by the devel...

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Activity and DNA contamination of commercial polymerase chain reaction reagents for the universal 16S rDNA real-time polymerase chain reaction detection of bacterial pathogens in blood

Cited by 85 publications

References 26 publications

First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

Diagnosis of prosthetic joint infections using UMD-Universal Kit and the automated multiplex-PCR Unyvero i60 ITI® cartridge system: a pilot study

Diagnosis of Bacteremia in Whole-Blood Samples by Use of a Commercial Universal 16S rRNA Gene-Based PCR and Sequence Analysis

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