title: Development and evaluation of a novel multicopy-element-targeting triplex PCR for detection of Mycobacterium avium subsp. paratuberculosis in feces.
abstract: The enteropathy called paratuberculosis (PTB), which mainly affects ruminants and has a worldwide distribution, is caused by Mycobacterium avium subsp. paratuberculosis. This disease significantly reduces the cost-effectiveness of ruminant farms, and therefore, reliable and rapid detection methods are needed to control the spread of the bacterium in livestock and in the environment. The aim of this study was to identify a specific and sensitive combination of DNA extraction and amplification to detect M. avium subsp. paratuberculosis in feces. Negative bovine fecal samples were inoculated with increasing concentrations of two different bacterial strains (field and reference) to compare the performance of four extraction and five amplification protocols. The best results were obtained using the JohnePrep and MagMax extraction kits combined with an in-house triplex real-time PCR designed to detect IS900, ISMap02 (an insertion sequence of M. avium subsp. paratuberculosis present in 6 copies per genome), and an internal amplification control DNA simultaneously. These combinations detected 10 M. avium subsp. paratuberculosis cells/g of spiked feces. The triplex PCR detected 1 fg of genomic DNA extracted from the reference strain K10. The performance of the robotized version of the MagMax extraction kit combined with the IS900 and ISMap02 PCR was further evaluated using 615 archival fecal samples from the first sampling of nine Friesian cattle herds included in a PTB control program and followed up for at least 4 years. The analysis of the results obtained in this survey demonstrated that the diagnostic method was highly specific and sensitive for the detection of M. avium subsp. paratuberculosis in fecal samples from cattle and a very valuable tool to be used in PTB control programs.
authors.affiliation: Departamento de Sanidad Animal, NEIKER-Tecnalia, Derio, Biscay, Spain isevilla@neiker.net.
authors.forename: Iker A
authors.identifier: http://orcid.org/0000-0003-3968-3390
authors.initials: IA
authors.lastname: Sevilla
authors.affiliation: Departamento de Sanidad Animal, NEIKER-Tecnalia, Derio, Biscay, Spain.
authors.forename: Joseba M
authors.initials: JM
authors.lastname: Garrido
authors.affiliation: Departamento de Sanidad Animal, NEIKER-Tecnalia, Derio, Biscay, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Molina
authors.affiliation: Departamento de Sanidad Animal, NEIKER-Tecnalia, Derio, Biscay, Spain.
authors.forename: María V
authors.initials: MV
authors.lastname: Geijo
authors.affiliation: Departamento de Sanidad Animal, NEIKER-Tecnalia, Derio, Biscay, Spain.
authors.forename: Natalia
authors.initials: N
authors.lastname: Elguezabal
authors.affiliation: Departamento de Sanidad Animal, NEIKER-Tecnalia, Derio, Biscay, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Vázquez
authors.affiliation: Departamento de Sanidad Animal, NEIKER-Tecnalia, Derio, Biscay, Spain.
authors.forename: Ramón A
authors.initials: RA
authors.lastname: Juste
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology*; D004251:DNA Transposable Elements; D005243:Feces / Q000382:microbiology*; D018628:Gene Dosage; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000302:isolation & purification*; D010283:Paratuberculosis / Q000175:diagnosis / Q000382:microbiology*
chemical_list: D004251:DNA Transposable Elements
references.citation: J Am Vet Med Assoc. 2005 Oct 15;227(8):1302-8
references.pmid: 16266021
references.citation: Appl Environ Microbiol. 2008 May;74(9):2751-8
references.pmid: 18326682
references.citation: BMC Microbiol. 2006 Oct 04;6:87
references.pmid: 17020599
references.citation: Vet Microbiol. 1995 Jul;45(2-3):211-7
references.pmid: 7571372
references.citation: Clin Diagn Lab Immunol. 2005 Jun;12(6):685-92
references.pmid: 15939741
references.citation: Vet Clin North Am Food Anim Pract. 2011 Nov;27(3):581-91, vi
references.pmid: 22023836
references.citation: J Clin Microbiol. 2006 Mar;44(3):1081-3
references.pmid: 16517898
references.citation: BMC Microbiol. 2008 Nov 25;8:204
references.pmid: 19032737
references.citation: Prev Vet Med. 2009 Jan 1;88(1):1-14
references.pmid: 18817995
references.citation: Vet Microbiol. 2007 Nov 15;125(1-2):36-48
references.pmid: 17582709
references.citation: Vet Clin North Am Food Anim Pract. 2011 Nov;27(3):525-35, v
references.pmid: 22023831
references.citation: J Appl Microbiol. 2009 Oct;107(4):1061-71
references.pmid: 19486426
references.citation: J Dairy Sci. 2012 Feb;95(2):618-29
references.pmid: 22281327
references.citation: Vet Microbiol. 2005 Feb 25;105(3-4):215-21
references.pmid: 15708818
references.citation: Proc Natl Acad Sci U S A. 2005 Aug 30;102(35):12344-9
references.pmid: 16116077
references.citation: J Clin Microbiol. 1993 Jun;31(6):1601-5
references.pmid: 8315002
references.citation: Vet Microbiol. 2000 Dec 20;77(3-4):387-98
references.pmid: 11118724
references.citation: BMC Res Notes. 2009 Nov 22;2:233
references.pmid: 19930604
references.citation: J Comp Pathol. 2005 Aug-Oct;133(2-3):184-96
references.pmid: 16045917
references.citation: PLoS One. 2013 May 28;8(5):e64568
references.pmid: 23724062
references.citation: Appl Environ Microbiol. 2011 Dec;77(24):8615-24
references.pmid: 22003015
references.citation: J Dairy Res. 2005 Nov;72(4):425-32
references.pmid: 16223457
references.citation: Foodborne Pathog Dis. 2009 May;6(4):513-8
references.pmid: 19415976
references.citation: Mol Cell Probes. 1998 Jun;12(3):133-42
references.pmid: 9664574
references.citation: Am J Vet Res. 2009 Apr;70(4):493-7
references.pmid: 19335105
references.citation: Crit Rev Microbiol. 2012 Feb;38(1):52-93
references.pmid: 22242906
references.citation: BMC Microbiol. 2007 Mar 12;7:18
references.pmid: 17352818
references.citation: J Vet Diagn Invest. 2013 Jan;25(1):27-34
references.pmid: 23166181