This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oravcová, K.
Right arrow Articles by Hernández, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oravcová, K.
Right arrow Articles by Hernández, M.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, February 2009, p. 445-450, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01442-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mycoplasma agalactiae p40 Gene, a Novel Marker for Diagnosis of Contagious Agalactia in Sheep by Real-Time PCR: Assessment of Analytical Performance and In-House Validation Using Naturally Contaminated Milk Samples{triangledown} ,{dagger}

Katarína Oravcová,1,2 Lorena López-Enríquez,1 David Rodríguez-Lázaro,3 and Marta Hernández1*

Laboratory of Molecular Biology and Microbiology,1 Food Safety and Technology Group, Instituto Tecnológico Agrario de Castilla y León (ITACyL), Valladolid, Spain,3 Food Research Institute, Bratislava, Slovakia2

Received 28 July 2008/ Returned for modification 27 October 2008/ Accepted 11 November 2008

We evaluated the capacity of the Mycoplasma agalactiae p40 gene as a diagnostic marker for contagious agalactia in sheep by quantitative real-time PCR. The p40 gene encodes an immunodominant adhesin that plays a key role in cytoadhesion of M. agalactiae. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent (GE), a quantification that is highly linear (R2 > 0.992) and efficient (PCR efficiency, >0.992) over a 6-log dynamic range, down to 10 GE. We evaluated the capacity of the assay to detect Mycoplasma agalactiae in 797 milk samples (373 raw sheep milk samples from refrigerated tanks of different farms and 424 milk samples from individual sheep of a flock positive for M. agalactiae). In parallel, we also tested the samples by using microbiological isolation coupled with microscopy identification and by a PCR method recommended by the World Organization for Animal Health. While our assay was able to detect 57 (15.28%) positive samples of the 373 milk samples from different farms, identification by microbiological isolation coupled with microscopy detected only 36 (9.65%) samples, and the conventional PCR detected 31 (8.31%) samples. These findings showed that our assay based on the p40 gene is more specific and sensitive for the detection of M. agalactiae in actual natural samples and, thus, can be a promising alternative tool for diagnosis and epidemiological studies of M. agalactiae infection.

* Corresponding author. Mailing address: Instituto Tecnológico Agrario de Castilla y León (ITACyL), Carretera de Burgos, km. 119, 47071 Valladolid, Spain. Phone: 34 983 317542. Fax: 34 983 410462. E-mail: ita-herperma{at}itacyl.es

{triangledown} Published ahead of print on 19 November 2008.

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/.

Journal of Clinical Microbiology, February 2009, p. 445-450, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01442-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»