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Pathogen quantitation in complex matrices : a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition
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Pontiroli, Alessandra, Travis, Emma Rachel, Sweeney, F. P., Porter, David, Gaze, William H., Mason, Sam, Hibberd, V., Holden, Jennifer, Courtenay, Orin and Wellington, E. M. H. (2011) Pathogen quantitation in complex matrices : a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition. PLoS One, Vol.6 (No.3). Article number e17916. doi:10.1371/journal.pone.0017916 ISSN 1932-6203.
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Official URL: http://dx.doi.org/10.1371/journal.pone.0017916
Abstract
Background: Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year.
Methodology/Principal Findings: We report a multi-operator blinded trial for a
rigorous comparison of five DNA extraction methods from a variety of soil and
faecal samples to assess recovery of M. bovis via real-time PCR detection. The
methods included four commercial kits: the QIAamp Stool Mini kit with a pretreatment
step, the FastDNA® Spin kit, the UltraCleanTM and PowerSoilTM soil kits
and a published manual method based on phenol:chloroform purification, termed
Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and
evaluated using a specific real-time PCR assay. A novel inhibition control assay was
used alongside spectrophotometric ratios to monitor the level of inhibitory
compounds affecting PCR, DNA yield, and purity. There were statistically
significant differences in M. bovis detection between methods of extraction and types
of environmental samples; no significant differences were observed between
operators. Processing times and costs were also evaluated. To improve M. bovis
detection further, the two best performing methods, FastDNA® Spin kit and
Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was
adopted, leading to improved sensitivities.
Conclusions: M. bovis was successfully detected in all environmental samples; DNA
extraction using FastDNA® Spin kit was the most sensitive method with highest
recoveries from all soil types tested. For troublesome faecal samples, we have used
and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25 x 105 cells g-1 using Griffiths and 4.25 x 106 cells g-1 using
FastDNA® Spin kit.
| Item Type: | Journal Article | ||||
|---|---|---|---|---|---|
| Subjects: | Q Science > QH Natural history > QH426 Genetics Q Science > QR Microbiology |
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| Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||
| Library of Congress Subject Headings (LCSH): | Mycobacterium bovis -- Detection, Bacteriology, Agricultural, Polymerase chain reaction, Genetics -- Technique | ||||
| Journal or Publication Title: | PLoS One | ||||
| Publisher: | Public Library of Science | ||||
| ISSN: | 1932-6203 | ||||
| Official Date: | 23 March 2011 | ||||
| Dates: |
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| Volume: | Vol.6 | ||||
| Number: | No.3 | ||||
| Article Number: | Article number e17916 | ||||
| DOI: | 10.1371/journal.pone.0017916 | ||||
| Status: | Peer Reviewed | ||||
| Publication Status: | Published | ||||
| Access rights to Published version: | Open Access (Creative Commons) | ||||
| Funder: | Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC), Great Britain. Dept. for Environment, Food & Rural Affairs (DEFRA) | ||||
| Grant number: | BB/E020925/1 (BBSRC), SE3231 (DEFRA) |
Data sourced from Thomson Reuters' Web of Knowledge
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