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

• Published in: PloS one Vol. 6; no. 11; p. e27369
• Main Authors: Travis, Emma R, Gaze, William H, Pontiroli, Alessandra, Sweeney, Francis P, Porter, David, Mason, Sam, Keeling, Matthew J C, Jones, Rebecca M, Sawyer, Jason, Aranaz, Alicia, Rizaldos, Elena Castellanos, Cork, Jennifer, Delahay, Richard J, Wilson, Gavin J, Hewinson, R Glyn, Courtenay, Orin, Wellington, Elizabeth M H
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 14.11.2011; Public Library of Science (PLoS)
• Subjects: Agriculture; Analysis; Animals; Assaying; Bacillus Calmette-Guerin vaccine; Bacteria; Badgers; BCG; Beef cattle; Biology; Bovidae; Cattle; Data processing; Data recovery; Deoxyribonucleic acid; Design optimization; Dilution; DNA; E coli; Epidemiology; Escherichia coli; Excretion; Experimental design; False Negative Reactions; Feces; Feces - microbiology; Health aspects; Infection; Infections; Laboratories; Latrines; Life sciences; Mammals; Medicine; Meles meles; Mustelidae - microbiology; Mycobacterium; Mycobacterium bovis; Mycobacterium bovis - genetics; Mycobacterium bovis - isolation & purification; Operators; Panels; Polymerase chain reaction; Population; Quantitative analysis; Real time; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - standards; Reliability analysis; Reproducibility; Science Policy; Studies; Sustainable development; Tuberculosis; Veterinary Science; Wildlife; Wildlife management; Zoonoses; United Kingdom > UK
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0027369

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.