Improved detection of microbial DNA after bead-beating before DNA isolation

Boer, Richard de; Peters, Remco P. H.; Gierveld, Sonja; Schuurman, T.; Kooistra-Smid, Mirjam; Savelkoul, Paul H. M.

doi:10.1016/j.mimet.2009.11.009

Cited by 100 publications

(78 citation statements)

References 15 publications

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“…The DNA extraction method used was modified from a previous method [10,19,21]. Consistent with previous studies [22,23], we found that the process of bead beating improves the efficiency of DNA isolation from culture strains (data not shown). Specific primers and probes for pan-dermatophyte and T. rubrum, which represent Fig.…”

Section: Discussionsupporting

confidence: 85%

Development and Evaluation of a Novel Real-Time PCR for Pan-Dermatophyte Detection in Nail Specimens

et al. 2015

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An accurate diagnosis of tinea unguium is necessary for the selection of antimycotics and successful treatment. To rapidly and accurately identify the aetiological agents causing tinea unguium, we improved upon the conventional boiling method for DNA extraction and developed a novel real-time PCR detection system that includes two assays. The two assays, based on the amplification of ribosomal internal transcribed spacer regions and 28S rDNA, were designed to detect pan-dermatophyte and Trichophyton rubrum, respectively. The analytical sensitivities of both assays permitted the detection of ten copies of plasmid DNA template. The analytical specificity of the detection system was confirmed using 11 dermatophyte strains and 25 non-dermatophyte strains. In total, 165 nail specimens were examined by microscopy, culture, conventional PCR, and the novel real-time PCR method. Real-time PCR gave positive results in 47.3 % of the specimens (78), a rate exceeding those obtained using microscopy (72, 43.6 %), conventional PCR (69, 41.8 %), and culture (49, 29.7 %). All conventional PCR-positive specimens were detected by real-time PCR, and real-time PCR detected nine specimens that were missed by conventional PCR. The results from latent class analysis, and further calculations, showed that real-time PCR was the most sensitive method, but the diagnostic specificity of the four approaches was equivalent. In particular, molecular approaches may be more effective than microscopy and culture when the clinical symptoms of tinea unguium are not evident.

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Section: Discussionsupporting

confidence: 85%

Development and Evaluation of a Novel Real-Time PCR for Pan-Dermatophyte Detection in Nail Specimens

et al. 2015

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“…It is well known that Gram-positive organisms (such as bifidobacteria) can be underrepresented in microbial profiling studies due to the presence of their thick cell wall [34]. Due to concern that poor representation of bifidobacteria from faecal samples may be due to difficulties in cell lysis during DNA extraction, we first assessed target sequence recovery from pure DNA mixtures.…”

Section: Discussionmentioning

confidence: 99%

Improved Detection of Bifidobacteria with Optimised 16S rRNA-Gene Based Pyrosequencing

et al. 2012

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The 16S rRNA gene is conserved across all bacteria and as such is routinely targeted in PCR surveys of bacterial diversity. PCR primer design aims to amplify as many different 16S rRNA gene sequences from as wide a range of organisms as possible, though there are no suitable 100% conserved regions of the gene, leading to bias. In the gastrointestinal tract, bifidobacteria are a key genus, but are often under-represented in 16S rRNA surveys of diversity. We have designed modified, ‘bifidobacteria-optimised’ universal primers, which we have demonstrated detection of bifidobacterial sequence present in DNA mixtures at 2% abundance, the lowest proportion tested. Optimisation did not compromise the detection of other organisms in infant faecal samples. Separate validation using fluorescence in situ hybridisation (FISH) shows that the proportions of bifidobacteria detected in faecal samples were in agreement with those obtained using 16S rRNA based pyrosequencing. For future studies looking at faecal microbiota, careful selection of primers will be key in order to ensure effective detection of bifidobacteria.

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“…Similarly, mycobacteria have a thick, waxy cell wall that is difficult to disrupt for the extraction of nucleic acids (9,17). High-energy mechanical disruption methods, such as sonication and bead beating, are commonly used for lysis of thickwalled organisms, since chemical, heat, freeze-thaw, or enzymatic lysis methods alone are less effective (1,11,22). Lysis protocols for mycobacteria have been reported that use lowenergy bead beating (2,6) in conjunction with heat or chemical or enzymatic lytic agents, which increase process complexity and potentially introduce PCR inhibitors.…”

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confidence: 99%

“…The mechanism of lysis by bead-beating has been attributed to high shear rates between beads and strong periodic vortical flow fields (13). The diameter of beads used during mechanical lysis is critical to lysis efficiency, with 100-m-diameter beads being more effective than larger-diameter beads at lysing Gram-positive bacteria (11,22).…”

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confidence: 99%

Mechanical Disruption of Lysis-Resistant Bacterial Cells by Use of a Miniature, Low-Power, Disposable Device

et al. 2011

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Molecular detection of microorganisms requires microbial cell disruption to release nucleic acids. Sensitive detection of thick-walled microorganisms such as Bacillus spores and Mycobacterium cells typically necessitates mechanical disruption through bead beating or sonication, using benchtop instruments that require line power. Miniaturized, low-power, battery-operated devices are needed to facilitate mechanical pathogen disruption for nucleic acid testing at the point of care and in field settings. We assessed the lysis efficiency of a very small disposable bead blender called OmniLyse relative to the industry standard benchtop Biospec Mini-BeadBeater. The OmniLyse weighs approximately 3 g, at a size of approximately 1.1 cm 3 without the battery pack. Both instruments were used to mechanically lyse Bacillus subtilis spores and Mycobacterium bovis BCG cells. The relative lysis efficiency was assessed through real-time PCR. Cycle threshold (C T ) values obtained at all microbial cell concentrations were similar between the two devices, indicating that the lysis efficiencies of the OmniLyse and the BioSpec Mini-BeadBeater were comparable. As an internal control, genomic DNA from a different organism was spiked at a constant concentration into each sample upstream of lysis. The C T values for PCR amplification of lysed samples using primers specific to this internal control were comparable between the two devices, indicating negligible PCR inhibition or other secondary effects. Overall, the OmniLyse device was found to effectively lyse tough-walled organisms in a very small, disposable, batteryoperated format, which is expected to facilitate sensitive point-of-care nucleic acid testing.

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Improved detection of microbial DNA after bead-beating before DNA isolation

Cited by 100 publications

References 15 publications

Development and Evaluation of a Novel Real-Time PCR for Pan-Dermatophyte Detection in Nail Specimens

Development and Evaluation of a Novel Real-Time PCR for Pan-Dermatophyte Detection in Nail Specimens

Improved Detection of Bifidobacteria with Optimised 16S rRNA-Gene Based Pyrosequencing

Mechanical Disruption of Lysis-Resistant Bacterial Cells by Use of a Miniature, Low-Power, Disposable Device

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