A comparative study evaluating the efficacy of IS_MAP04 with IS900 and IS_MAP02 as a new diagnostic target for the detection of Mycobacterium avium subspecies paratuberculosis from bovine faeces

Authors

Marcel de Kruijf, Department of Biological Sciences, Cork Institute of Technology, Cork, Ireland
Rodney Govender, Department of Biological Sciences, Cork Institute of Technology, Cork, Ireland
Dermot Yearsley, Department of Agriculture and Food Veterinary Laboratory Service Ireland
Aidan Coffey, Department of Biological Sciences, Cork Institute of Technology, Cork, IrelandFollow
Jim O'Mahony, Department of Biological Sciences, Cork Institute of Technology, Cork, IrelandFollow

ORCID

https://orcid.org/0000-0003-4116-9913

Document Type

Article

Disciplines

Biology

Publication Details

Veterinary Microbiology

© 2017 Elsevier Ltd

Abstract

The aim of this study was to investigate the efficacy of IS_MAP04 as a potential new diagnostic quantitative PCR (qPCR) target for the detection of Mycobacterium avium subspecies paratuberculosis from bovine faeces. IS_MAP04 primers were designed and tested negative against non-MAP strains. The detection limit of IS_MAP04 qPCR was evaluated on different MAP K-10 DNA concentrations and on faecal samples spiked with different MAP K-10 cell dilutions. A collection of 106 faecal samples was analysed and the efficacy of IS_MAP04 was statistically compared with IS900 and IS_MAP02. The detection limits observed for IS_MAP04 and IS900 on MAP DNA was 34 fg and 3.4 fg respectively. The detection limit of MAP from inoculated faecal samples was 102 CFU/g for both IS_MAP04 and IS900 targets and a detection limit of 102 CFU/g was also achieved with a TaqMan qPCR targeting IS_MAP04. The efficacy of IS_MAP04 to detect positive MAP faecal samples was 83.0% compared to 85.8% and 83.9% for IS900 and IS_MAP02 respectively. Strong kappa agreements were observed between IS_MAP04 and IS900 (κ = 0.892) and between IS_MAP04 and IS_MAP02 (κ = 0.897). As a new molecular target, IS_MAP04 showed that the detection limit was comparable to IS900 to detect MAP from inoculated faecal material. The MAP detection efficacy of IS_MAP04 from naturally infected faecal samples proved to be relatively comparable to IS_MAP02, but yielded efficacy results slightly less than IS900. Moreover, IS_MAP04 could be of significant value when used in duplex or multiplex qPCR assays.

Recommended Citation

De Kruijf, M. et al., 2017. A comparative study evaluating the efficacy of IS_MAP04 with IS900 and IS_MAP02 as a new diagnostic target for the detection of Mycobacterium avium subspecies paratuberculosis from bovine faeces. Veterinary Microbiology, 204, pp.104–109. Available at: http://dx.doi.org/10.1016/j.vetmic.2017.04.020.