Pontiroli, Alessandra, Travis, Emma Rachel, Sweeney, F. P., Porter, David, Gaze, William H., Mason, Sam (Sam A.), Hibberd, V. (Victoria), Holden, Jennifer, Courtenay, Orin and Wellington, E. M. H. (Elizabeth M. H.), 1954-. (2011) Pathogen quantitation in complex matrices : a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition. PL o S One, Vol.6 (No.3). e17916. ISSN 1932-6203

Preview	PDF WRAP_Wellington_0380313-lf-170511-pontiroli_cc_revised_2.pdf - Draft Version - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader Download (316Kb)
Preview	Image (TIFF) (Figure 2) WRAP_Wellinton_Fig2.tiff - Draft Version Download (382Kb)
Preview	Image (TIFF) (Figure 3) WRAP_Wellintgon_Fig_3_rev.tif - Draft Version Download (797Kb)
Preview	Image (TIFF) (Figure 4) WRAP_WEllington_Fig_4_rev.tif - Draft Version Download (364Kb)
Preview	Image (TIFF) (Figure 5) WRAP_Wellintgon_Fig5.tif - Draft Version Download (264Kb)
	Microsoft Excel (Table S1) WRAP_Wellinton_TableS1.xlsx - Draft Version Download (9Kb)
	Microsoft Excel (Table S2) WRAP_Wellington_TableS2.xlsx - Draft Version Download (35Kb)

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
Divisions:	Faculty of Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH):	Mycobacterium bovis -- Detection, Bacteriology, Agricultural, Polymerase chain reaction, Genetics -- Technique
Journal or Publication Title:	PL o S One
Publisher:	Public Library of Science
ISSN:	1932-6203
Date:	23 March 2011
Volume:	Vol.6
Number:	No.3
Page Range:	e17916
Identification Number:	10.1371/journal.pone.0017916
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
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)
References:	1. Young JS, Gormley E, Wellington EM (2005) Molecular detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in soil. Appl Environ Microbiol 71: 1946-1952. 2. Sweeney FP, Courtenay O, Ul-Hassan A, Hibberd V, Reilly LA, et al. (2006) Immunomagnetic recovery of Mycobacterium bovis from naturally infected environmental samples. Lett Appl Microbiol 43: 364-369. 3. Donnelly CA, Woodroffe R, Cox DR, Bourne J, Gettinby G, et al. (2003) Impact of localized badger culling on tuberculosis incidence in British cattle. Nature 426: 834- 837. 4. Coleman JD, Coleman MC, Warburton B (2006) Trends in the incidence of tuberculosis in possums and livestock, associated with differing control intensities applied to possum populations. New Zealand Vet J 54: 52-60. 5. Caley P, Hickling GJ, Cowan PE, Pfeiffer DU (1999) Effects of sustained control of brushtail possums on levels of Mycobacterium bovis infection in cattle and brushtail possum populations from Hohotaka, New Zealand. New Zealand VetJ 47: 133-142. 6. Dokoupil S (1964) Survival of M. tuberculosis in grass, soil, bedding in cow sheds and urine. Vedecke Prace Vyzkumneho Ustavu Veterinarniho Lekarstvi v Brne 3: 49- 52. 7. Genov I (1965) The effects of certain physical and chemical agents on Mycobacterium tuberculosis. Veteriární Medicina Nauki Sofia 2: 97–107. 8. Maddock ECG (1933) Studies on the survival time of the bovine tubercle bacillus in soil, soil and dung, in dung and on grass, with experiments on the preliminary treatment of infected organic matter and the cultivation of the organism. Epidem Infect 33: 103-117. 9. Williams RS, Hoy WA (1930) The viability of B. tuberculosis (bovinus) on pasture land, in stored faeces and in liquid manure. J Hygiene 30: 413-419. 10. Young JS, Gormley E, Wellington EMH (2005) molecular detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in soil. Appl Environ Microbiol 71: 1946-1952. 11. Courtenay O, Reilly LA, Sweeney FP, Hibberd V, Bryan S, et al. (2006) Is Mycobacterium bovis in the environment important for the persistence of bovine tuberculosis? Biol Letters 2: 460-462. 12. Courtenay O, Wellington EMH (2008) Mycobacterium bovis in the environment: towards our understanding of its biology. BCVA Cattle Practice 16: 122-126. 13. Reilly LA, Courtenay O (2007) Husbandry practices, badger sett density and habitat composition as risk factors for transient and persistent bovine tuberculosis on UK cattle farms. Prev Vet Medicine 80: 129-142. 14. Hiroshi A, Akiko I, Eiji A, Sou-ichi M, Yumiko O, et al. (2007) Gene expression profile of Vibrio cholerae in the cold stress-induced viable but non-culturable state. Environ Microbiol 9: 869-879. 15. Sweeney FP, Courtenay O, Hibberd V, Hewinson RG, Reilly LA, et al. (2007) Environmental monitoring of Mycobacterium bovis in badger feces and badger sett soil by real-time PCR, as confirmed by immunofluorescence, immunocapture, and cultivation. Appl Environ Microbiol 73: 7471-7473. 16. Lantz PG, Matsson M, Wadström T, Rådström P (1997) Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR. J Microbiol Meth 28: 159-167. 17. Ghosh J, Larsson P, Singh B, Pettersson BMF, Islam NM, et al. (2009) Sporulation in mycobacteria. Proc Natl Acad Sci USA 106: 10781-10786. 25 18. Tsai YL, Olson BH (1991) Rapid method for direct extraction of DNA from soil and sediments. Appl Environ Microbiol 57: 1070-1074. 19. Griffiths RI, Whiteley AS, O'Donnell AG, Bailey MJ (2000) Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA and rRNA-based microbial community composition. Appl Environ Microbiol 66: 5488-5491. 20. Dong D, Yan A, Liu H, Zhang X, Xu Y (2006) Removal of humic substances from soil DNA using aluminium sulfate. J Microbiol Meth 66: 217-222. 21. Sagova-Mareckova M, Cermak L, Novotna J, Plhackova K, Forstova J, et al. (2008) Innovative methods for soil DNA purification tested in soils with widely differing characteristics. Appl Environ Microbiol 74: 2902-2907. 22. Purdy KJ, Embley TM, Takii S, Nedwell DB (1996) rapid extraction of dna and rrna from sediments by a novel hydroxyapatite spin-column method. Appl Environ Microbiol 62: 3905-3907. 23. Obika S, Nanbu D, Hari Y, Andoh J, Mori OK, et al. (1998) Stability and structural features of the duplexes containing nucleoside analogues with a fixed N type conformation, 2‘-O,4‘-C-methyleneribonucleosides. Tetrahedron Lett 39: 5401- 5404. 24. Simeonov A, Nikiforov T (2002) Single nucleotide polymorphism genotyping using short, fluorescently labelled locked nucleic acid (LNA) probes and fluorescence polarisation detection. Nucleic Acid Res 30: e91. 25. Holland BS, Copenhaver MD (1987) An improved sequentially rejective Bonferroni Test procedure. Biometrics 43: 417-423. 26. Artz RR, Avery LM, Jones DL, Killham K (2006) Potential pitfalls in the quantitative molecular detection of Escherichia coli O157:H7 in environmental matrices. Can J Microbiol 52: 482-488. 27. Kaser M, Ruf MT, Hauser J, Marsollier L, Pluschke G (2009) Optimized method for preparation of DNA from pathogenic and environmental mycobacteria. Appl Environ Microbiol 75: 414-418. 28. Chen H, Rangasamy M, Tan SY, Wang H, Siegfried BD (2010) Evaluation of five methods for total DNA Extraction from western corn rootworm beetles. PLoS ONE 5(8): e11963. doi:10.1371/journal.pone.0011963. 29. Aldous WK, Pounder JI, Cloud JL, Woods GL (2005) Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR. J Clin Microbiol 43: 2471-2473. 30. Whitehouse CA, Hottel HE (2007) Comparison of five commercial DNA extraction kits for the recovery of Francisella tularensis DNA from spiked soil samples. Mol Cell Probes 21: 92-96. 31. Queipo-Ortuño M, Tena F, Colmenero J, Morata P (2008) Comparison of seven commercial DNA extraction kits for the recovery of Brucella DNA from spiked human serum samples using real-time PCR. Eur J Clin Microbiol Infect Dis 27: 109- 114. 32. Durnez L, Stragier P, Roebben K, Ablordey A, Leirs H, et al. (2009) A comparison of DNA extraction procedures for the detection of Mycobacterium ulcerans, the causative agent of Buruli ulcer, in clinical and environmental specimens. J Microbiol Meth 76: 152-158. 33. Inoue M, Tang W, Wee S, Barkham T (2010) Audit and improve! Evaluation of a real-time probe-based PCR assay with internal control for the direct detection of Mycobacterium tuberculosis complex. Eur J Clinical Microbiol Infect Dis: 1-5. 34. Hartman LJ, Coyne SR, Norwood DA (2005) Development of a novel internal positive control for Taqman® based assays. Mol Cell Probes 19: 51-59. 26 35. Dineen SM, Aranda R, Anders DL, Robertson JM (2010) An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil. J Appl Microbiol 109: 1886-1896.
URI:	http://wrap.warwick.ac.uk/id/eprint/4547

Data sourced from Thomson Reuters' Web of Knowledge

Request changes to a record

Actions (login required)

View Item