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Development of a specific immunomagnetic capture-PCR for rapid detection of viable Mycoplasma agalactiae in sheep milk samples

Sanna, Giovanna and Lecca, Valentina and Foddai, Antonio and Tola, Sebastiana (2014) Development of a specific immunomagnetic capture-PCR for rapid detection of viable Mycoplasma agalactiae in sheep milk samples. Journal of applied microbiology, Vol. 117 (6), p. 1585-1591. eISSN 1364-5072. Article.

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DOI: 10.1111/jam.12657

Abstract

Aims: To develop an immunomagnetic capture (IMC) to detect viable Mycoplasma agalactiae in routine ovine milk samples.
Methods and Results: Polyclonal antibodies against two M. agalactiae membrane surface proteins (P80 and P55) were covalently conjugated to magnetic beads (MBs) to form MB-Ab80 and MB-Ab55. Mycoplasma agalactiae cells were captured by a specific antigen–antibody reaction and magnetic separation. Immunomagnetic capture (IMC) was used to isolate and concentrate M. agalactiae in serial decimal dilutions and in artificially contaminated milk to facilitate subsequent detection by PCR. A 375-bp fragment of M. agalactiae was amplified using a pair of M. agalactiae-specific primers in PCR. The limit of detection of IMC-PCR method ranged from 10 to 102 CCU ml -1 when mycoplasmas were resuspended in PBS and from 102 to 103 CCU ml-1 when mycoplasmas were resuspended in uncontaminated ovine milk. This study also describes the application of IMC-PCR method to test for M. agalactiae in 516 milk samples collected from sheep with suspected contagious agalactia. Its performance was evaluated relative to culture.
Conclusions: This report has demonstrated for the first time, the effective use of rapid and reliable IMC combined with PCR assay for the detection of viable M. agalactiae.
Significance and Impact of the Study: The method IMC-PCR provides an alternative to conventional microbiological detection, method and it could be applied to quick detection of M. agalactiae in routine sheep milk samples.

Item Type:Article
ID Code:10288
Status:Published
Refereed:Yes
Uncontrolled Keywords:Antibodies, immunocapture, membrane surface proteins, Mycoplasma agalactiae, PCR
Subjects:Area 07 - Scienze agrarie e veterinarie > VET/06 Parassitologia e malattie parassitarie degli animali
Divisions:002 Altri enti e centri di ricerca del Nord Sardegna > Istituto zooprofilattico sperimentale della Sardegna, Sassari
Publisher:Blackwell / Wiley
eISSN:1364-5072
Copyright Holders:© 2014 The Authors published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology
Deposited On:10 Nov 2014 17:44

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