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An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of mycobacterium bovis

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Travis, Emma Rachel, Gaze, William H., Pontiroli, Alessandra, Sweeney, Francis P., Porter, David, Mason, Sam (Sam A.), Keeling, Matthew James, Jones, Rebecca M., Sawyer, Jason, Aranaz, Alicia, Castellanos Rizaldos, Elena, Cork, Jennifer, Delahay, Richard J., Wilson, Gavin J., Hewinson, R. Glyn, Courtenay, Orin and Wellington, E. M. H. (Elizabeth M. H.), 1954-. (2011) An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of mycobacterium bovis. PLoS One, Vol.6 (No.11). e27369. ISSN 1932-6203

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Official URL: http://dx.doi.org/10.1371/journal.pone.0027369

Abstract

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.

Item Type:	Journal Article
Subjects:	Q Science > QR Microbiology
Divisions:	Faculty of Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH):	Mycobacterium bovis, Polymerase chain reaction, Bacteria -- Identification
Journal or Publication Title:	PLoS One
Publisher:	Public Library of Science
ISSN:	1932-6203
Date:	2011
Volume:	Vol.6
Number:	No.11
Page Range:	e27369
Identification Number:	10.1371/journal.pone.0027369
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Open Access
Funder:	Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC), Great Britain. Dept. for Environment, Food & Rural Affairs (DEFRA)
Grant number:	BBSRCBB/E020925/1 (BBSRC), SE3231 (DEFRA)
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URI:	http://wrap.warwick.ac.uk/id/eprint/41971