Real-Time PCR-Based Identification of Bacterial and Fungal Pathogens from Blood Samples

Mai, Madeleine; Müller, Iris; Maneg, Daniela; Lohr, Benedikt; Haecker, Achim; Haberhausen, Gerd; Hunfeld, Klaus-Peter

doi:10.1007/978-1-4939-1776-1_14

Cited by 8 publications

(5 citation statements)

References 15 publications

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“…Thus rapid methods are viewed as alternative means to detect and monitor Cronobacter spp. Nucleic acid amplification methods including PCR (Moraes and Maruniak 1997 ; Chen et al 2015 ), multiplex PCR (Gordon et al 2015 ; Zhang et al 2015 ), real-time PCR (Cecilia et al 2015 ; Mai et al 2015 ) have attracted the attention of a plethora of engineers and scientists due to their relatively specific and accurate characteristics (Tafelski et al 2015 ), but the limitations of their thermal cycling, complicacy and low level sensitivity impedes its applicability in rapid and routine monitoring of pathogens (Cornelissen et al 2016 ). An easy method to operate loop-mediated isothermal amplification (LAMP) developed by Notomi in 2000 showed attractive potential and sufffered the problems common in complicated reactions with the inhibitory components extracted from the crude samples (Notomi et al 2000 ).…”

Section: Introductionmentioning

confidence: 99%

Probe-free label system for rapid detection of Cronobacter genus in powdered infant formula

Jiang

et al. 2018

AMB Expr

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Cronobacter species previously known as Enterobacter sakazakii poses high risks to neonates and infants. In this work a rapid detection method was developed which combined loop-mediated isothermal amplification with lateral flow assay for detection of Cronobacter species in powdered infant formula. The fast amplification reaction without betaine was established and capable of performing DNA replication within 25 min. Based on the novel probe-free labeling methods, we established a lateral flow assay to capture the specific loop-mediated isothermal amplification amplicons which were labeled with fluorescein isothiocyanate and biotin. And the final detection time of this system was within 40 min. The false positive results of the lateral flow assay induced by primer dimer tagged with fluorescein isothiocyanate and biotin were eliminated by Taq single strand DNA binding protein (4 ng/μL). Simultaneously, the efficiency of the fast loop-mediated isothermal amplification assay was achieved. By injection of Taq SSB into the amplification assay as a replacement for betaine, the novel probe-free method could detect Cronobacter species with high specificity and sensitivity at the detection limit in PIF of 101 cfu/g. Our overall strategy has excellent potential in the rapid diagnosis of Cronobacter species label-free by integrating loop-mediated isothermal amplification and lateral flow assay.

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Section: Introductionmentioning

confidence: 99%

Probe-free label system for rapid detection of Cronobacter genus in powdered infant formula

Jiang

et al. 2018

AMB Expr

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“…New bacterial identification approaches based on advanced genotypic and mass spectrometric techniques have been developed. − Genotypic methods include high throughput sequencing, 16S rRNA gene sequencing, and PCR. − However, these methods suffer from complexity in sample pretreatment and expensive kits, limiting their widespread in clinical usages. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) profiling of bacteria has been exploited since the 1990s. − Identification is based on spectra pattern matching between the mass spectra of samples and a library of standard mass spectra of known bacteria.…”

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confidence: 99%

Bacterial Whole Cell Typing by Mass Spectra Pattern Matching with Bootstrapping Assessment

et al. 2017

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Bacterial typing is of great importance in clinical diagnosis, environmental monitoring, food safety analysis, and biological research. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is now widely used to analyze bacterial samples. Identification of bacteria at the species level can be realized by matching the mass spectra of samples against a library of mass spectra of known bacteria. Nevertheless, in order to reasonably type bacteria, identification accuracy should be further improved. Herein, we propose a new framework to the identification and assessment for MALDI-MS based bacterial analysis. Our approach combines new measures for spectra similarity and a novel bootstrapping assessment. We tested our approach on a general data set containing the mass spectra of 1741 strains of bacteria and another challenging data set containing 250 strains, including 40 strains in the Bacillus cereus group that were previously claimed to be impossible to resolve by MALDI-MS. With the bootstrapping assessment, we achieved much more reliable predictions at both the genus and species level, and enabled to resolve the Bacillus cereus group. To the best of the authors' knowledge, our method is the first to provide a statistical assessment to MALDI-MS based bacterial typing that could lead to more reliable bacterial typing.

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“…It should be noted that, commonly used approaches for bacterial identification include the traditional plate method and molecular analytical biotechniques, such as PCR, mass spectrometry, and gene sequencing. − The plate method is considered as a golden standard for culturable bacteria. , However, it still requires the assistance of molecular analytical techniques to identify the specific bacteria species. Initially, we were planning to develop a microfluidics-based single or multiplex real-time PCR to identify the captured bacteria mixture.First of all, cross-reactivity or interference was observed in the simultaneous amplification process due to the presence of multiple pairs of primers and probes, which led to unreliable results.…”

Section: Resultsmentioning

confidence: 99%

Microfluidic Air Sampler for Highly Efficient Bacterial Aerosol Collection and Identification

Bian

Wang

et al. 2016

Anal. Chem.

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The early warning capability of the presence of biological aerosol threats is an urgent demand in ensuing civilian and military safety. Efficient and rapid air sample collection in relevant indoor or outdoor environment is a key step for subsequent analysis of airborne microorganisms. Herein, we report a portable battery-powered sampler that is capable of highly efficient bioaerosol collection. The essential module of the sampler is a polydimethylsiloxane (PDMS) microfluidic chip, which consisted of a 3-loop double-spiral microchannel featuring embedded herringbone and sawtooth wave-shaped structures. Vibrio parahemolyticus (V. parahemolyticus) as a model microorganism, was initially employed to validate the bioaerosol collection performance of the device. Results showed that the sampling efficacy reached as high as >99.9%. The microfluidic sampler showed greatly improved capturing efficiency compared with traditional plate sedimentation methods. The high performance of our device was attributed to the horizontal inertial centrifugal force and the vertical turbulence applied to airflow during sampling. The centrifugation field and turbulence were generated by the specially designed herringbone structures when air circulated in the double-spiral microchannel. The sawtooth wave-shaped microstructure created larger specific surface area for accommodating more aerosols. Furthermore, a mixture of bacterial aerosols formed by V. parahemolyticus, Listeria monocytogenes, and Escherichia coli was extracted by the microfluidic sampler. Subsequent integration with mass spectrometry conveniently identified the multiple bacterial species captured by the sampler. Our developed stand-alone and cable-free sampler shows clear advantages comparing with conventional strategies, including portability, easy-to-use, and low cost, indicating great potential in future field applications.

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Real-Time PCR-Based Identification of Bacterial and Fungal Pathogens from Blood Samples

Cited by 8 publications

References 15 publications

Probe-free label system for rapid detection of Cronobacter genus in powdered infant formula

Probe-free label system for rapid detection of Cronobacter genus in powdered infant formula

Bacterial Whole Cell Typing by Mass Spectra Pattern Matching with Bootstrapping Assessment

Microfluidic Air Sampler for Highly Efficient Bacterial Aerosol Collection and Identification

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