Improved methods for the detection of Bacillus anthracis spores by the polymerase chain reaction

Johns, Malcolm B.; Harrington, L.; Titball, Richard W.; Leslie, Dario L.

doi:10.1111/j.1472-765x.1994.tb00856.x

Cited by 29 publications

(27 citation statements)

References 5 publications

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“…Bacillus spores carry extracellular DNA that may be bound on the bacterial spore coats. This extracellular DNA is not readily removed by multiple wash steps and can be amplified during PCR even without mechanical lysis (5,19). Although it is possible that extracellular DNA was coamplified in the experiments described here, a control experiment demonstrated that mechanical lysis significantly increased (between 10-and 100-fold) the amount of PCR-amplifiable target DNA, a finding in line with the expectation that mechanical lysis is required to achieve a low limit of detection.…”

Section: Discussionsupporting

confidence: 64%

“…Having BSA present in the master mix was found to be important for obtaining robust and reproducible experimental results when zirconia/silica beads were used in either device, especially at lower BCG cell concentrations, as dis- B. subtilis spores are known to carry extracellular DNA (5,19). To rule out the possibility that PCR experiments predominantly detected extracellular DNA, we conducted a control experiment.…”

Section: Vol 49 2011 Mechanical Pathogen Lysis Using a Miniaturizedmentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 64%

Section: Vol 49 2011 Mechanical Pathogen Lysis Using a Miniaturizedmentioning

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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“…Viable spores are not the only source of DNA; damaged (nonviable) spores and vegetative cells (or fragments) will contain DNA that may also be present. Extreme conditions must be employed to release the DNA from spores (Marmur 1961; Johns et al. 1994; Belgrader et al.…”

Section: Introductionmentioning

confidence: 99%

Bacillus anthracis spore suspensions: determination of stability and comparison of enumeration techniques

2008

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Aim: To determine the stability and variability in concentration of spore suspensions of Bacillus anthracis (BA) spore suspensions by comparing different methods of enumeration and to detect changes, if any, under different storage conditions. Methods and Results: Plate and microscope counts were compared to measuring the genomic equivalents based on DNA content BA spore suspensions. We developed chemical methods to extract spore DNA and extra‐spore (ES) DNA. DNA mass was determined by gel electrophoresis and QPCR assays were developed using the markers on the chromosome (rpoB) and the pXO1 plasmid (pag). The plate counts and microscope counts were very stable (for up to 900 days). The effect of freezing and the presence of additives in samples were tested for up to 300 days, and the results indicated that the additives tested and freezing did not decrease the viability or microscope counts. Conclusions: Bacillus anthracis spore suspensions can be stored for long periods of time without significant loss of viability or clumping. The content of ES DNA was variable and changed with time. Significant and Impact of the Study: The study shows that BA spore suspensions can be developed for reference materials providing a uniform basis for comparing detection equipment and results from different laboratories.

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“…Since the aim was to detect both vegetative cells and spores, another question was which treatment would be strong enough to release DNA from spores but not so strong that it destroys the DNA released from vegetative [24,43], sonication [10], mechanical breaking (cell milling) [28] and germination with subsequent lysis of vegetative cells [28]. In our study, mechanical lysing for 3 min gave the same yield of DNA, measured by real-time PCR, for both vegetative cells and spores in pure cultures.…”

Section: Discussionmentioning

confidence: 99%

Detection of Bacillus cereus group bacteria from cardboard and paper with real-time PCR

Priha

Hallamaa

Saarela

et al. 2004

J IND MICROBIOL BIOTECHNOL

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The aim of this study was to develop a PCR-based rapid method to detect Bacillus cereus group cells from paper and cardboard. Primers targeting the 16S rDNA and real-time PCR with SYBR green I detection were used in order to be able to also quantify the target. Both autoclaved cardboard samples spiked with B. cereus vegetative cells or spores and naturally contaminated paper and cardboard samples were studied. Results were compared with culturing verified by commercial (API) tests. Several different methods were tested for DNA isolation from the paper and cardboard samples. Two commercial kits intended for soils, the UltraClean soil DNA kit and the FastDNA spin kit for soil, gave the most reproducible results. In spiked samples, the average yield was 50% of added vegetative cells, but spore yield was only about 10%. PCR results from adding vegetative cells correlated with added colony-forming unit (cfu) values ( r=0.93, P <0.001) in the range 100-10,000 cfu g(-1). Three out of nine studied paper and cardboard samples contained B. cereus group bacteria, based both on culturing and real-time PCR. The numbers were 10(2)-10(3) bacteria g(-1); and PCR gave somewhat higher results than culturing. Thus, real-time PCR can be used as a rapid semi-quantitative method to screen paper and cardboard samples for contamination with B. cereus group bacteria.

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Improved methods for the detection of Bacillus anthracis spores by the polymerase chain reaction

Cited by 29 publications

References 5 publications

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

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

Bacillus anthracis spore suspensions: determination of stability and comparison of enumeration techniques

Detection of Bacillus cereus group bacteria from cardboard and paper with real-time PCR

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