Truncated Human Cytidylate-Phosphate-Deoxyguanylate-Binding Protein for Improved Nucleic Acid Amplification Technique-Based Detection of Bacterial Species in Human Samples

Sachse, Svea; Straube, Eberhard; Lehmann, Marc; Bauer, Michael; Rußwurm, Stefan; Schmidt, Karl‐Hermann

doi:10.1128/jcm.02242-08

Cited by 48 publications

(36 citation statements)

References 30 publications

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“…This can be very challenging if genomic DNA is copurified with targeted microbial DNA (11)(12)(13). A number of solutions to this problem have been proposed (14)(15)(16)(17)(18)(19) but, to date, none have achieved the desired sensitivity (20). It should be noted that many of these methods do appear to capture a significantly higher number of total positives than culture, and the "extra" detections are often correlated with clinical indications of bloodstream infection; however, an inability to capture all positives detected by culture (false negatives) has been an issue.…”

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confidence: 99%

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

et al. 2014

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The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml wholeblood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.

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confidence: 99%

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

et al. 2014

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“…Fungal DNA was enriched via a specific binding matrix based on DNA affinity chromatography using a matrix-immobilized DNA binding protein (Looxster; SIRS Lab) which recognizes characteristic motifs within fungal DNA. When a complex DNA mixture is applied onto the matrix, more than 90% of the human background DNA is removed by two wash steps (9), altering the final ratio of fungal DNA to human DNA, potentially increasing the sensitivity of the downstream protocols.…”

Section: Methodsmentioning

confidence: 99%

“…This has been achieved mainly by targeting multicopy genes, such as the ribosomal gene cluster, and with the use of new technology, such as molecular-beacon-based assays or assays based on nucleic acid sequence-based amplification (10)(11)(12). A novel protocol for selective pathogen DNA enrichment based on affinity purification has successfully been used for the diagnosis of sepsis (9). This protocol is based on affinity purification of unmethylated microbial DNA due to selective binding to a protein.…”

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confidence: 99%

Pathogen-Specific DNA Enrichment Does Not Increase Sensitivity of PCR for Diagnosis of Invasive Aspergillosis in Neutropenic Patients

et al. 2011

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PCR assays designed for the diagnosis of invasive aspergillosis (IA) in high-risk patients have to detect minute amounts of target DNA to reach sufficient analytical sensitivity to be of clinical use. This prospective study assessed the use of a novel strategy for selective pathogen DNA enrichment for enhancing the performance of diagnostic PCR in a direct comparison with a highly sensitive in-house quantitative PCR (qPCR) assay and the galactomannan enzyme-linked immunosorbent assay (ELISA). Surprisingly, and in contrast to experience with other patient groups, the novel protocol for selective pathogen DNA enrichment did not enhance but instead significantly impaired sensitivity. This could be explained by the small amounts of host DNA in the specimens, which were derived mostly from severely neutropenic patients. In the qPCR assay, positive samples required an average of 43.5 amplification cycles (range, 39.2 to 50) for detection in the in-house PCR. Repetitive testing of selected samples showed test positivity to be variable, most likely due to the small amounts of target DNA. Despite this, the in-house protocol proved helpful in the diagnosis of IA, detecting 2 out of 3 patients with probable IA and 10 out of 19 patients with possible IA. Our results underline the necessity for diagnostic PCR protocols that help diagnose IA to be highly sensitive and show that selective pathogen DNA enrichment using affinity purification may not be useful in severely neutropenic patients.

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“…Additionally, some reports have shown a co-amplified background signal due to unspecific primer binding [125,127]. Due to the excess of human DNA in clinical samples [128,129], the large background nucleic acid can hamper the efficiency of the SDA considerably.…”

Section: Sda: Strand Displacement Amplificationmentioning

confidence: 99%

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Tröger¹,

Niemann²,

Gärtig³

et al. 2015

J Nanomed Nanotechnol

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Nucleic acid amplification technologies (NAATs) offer the most sensitive tests in the clinical laboratory. These techniques are used as a powerful tool for screening and diagnosis of infectious diseases. Isothermal methods, as an alternative to polymerase chain reaction (PCR), require no thermocycling machine and can mostly be performed with reduced time, high throughput, and accurate and reliable results.However, current molecular diagnostic approaches generally need manual analysis by qualified and experienced personal which is a highly complex, time-consuming and labor-intensive task. Thus, the demand for simpler, miniaturized systems and assays for pathogen detection is steadily increasing. Microfluidic platforms and lab-on-a-chip devices have many advantages such as small sample volume, portability and rapid detection time and enable point-of-care diagnosis.In this article, we review several isothermal amplification methods and their implementation in microsystems in relation to quantification of nucleic acids. miniaturized systems can be diverse. The majority of publications are focussed on clinical diagnostics including foodborne organisms for environmental monitoring [10][11][12]. In case of an infectious disease, cultivation and phenotypic characterization are still the standard methods of microbiologists which need days up to weeks to obtain a diagnostic result. Hence, scientists started to make use of nucleic acid amplification methods to accelerate the analysis. The most widespread and well known technique for this purpose is the polymerase chain reaction (PCR) [10,13], which exploits the activity of the DNA polymerase. The method is based on a thermal cycling process consisting of repeated cycles of heating and cooling steps allowing for DNA melting, sequence specific primer binding and enzymatic amplification of the target nucleic acid. The quantitative assessment of target copies is possible by using a real-time PCR approach, where a concentration dependent signal (e.g. fluorescence) increases over time [14]. The latter enables the user to easily assess the pathogen load of patient samples [15][16][17][18]. Since the first example of a PCR on a miniaturized system in 1994 [19,20], numerous devices were presented, which integrate not just the amplification, but also sample preparation and detection steps [9,[21][22][23][24][25][26][27][28]. Just a few of those microsystems have reached the market so far, but some have shown a fascinating potential to replace the classical laboratory-oriented state-of-the art [29][30][31][32][33]. setups has been shown to obtain a similar result [34]. A smaller heat capacity allows for rapid changes in temperature beneficial for the PCR time as well as a higher parallelism of multiple genetic samples [34].However, a major drawback of the integration of PCR to microsystems is the necessity of sophisticated instrumentation. The reaction requires a thermal cycling instrumentation, space and considerable expertise [35]. Therefore, isothermal reactions for the ta...

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Truncated Human Cytidylate-Phosphate-Deoxyguanylate-Binding Protein for Improved Nucleic Acid Amplification Technique-Based Detection of Bacterial Species in Human Samples

Cited by 48 publications

References 30 publications

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

Pathogen-Specific DNA Enrichment Does Not Increase Sensitivity of PCR for Diagnosis of Invasive Aspergillosis in Neutropenic Patients

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

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