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Duplex real-time PCR assay for rapid identification of Staphylococcus aureus isolates from dairy cow milk

Published online by Cambridge University Press:  12 March 2013

Rachel Pilla ,
Gustavo G M Snel ,
Michela Malvisi  and
Renata Piccinini
Rachel Pilla
Affiliation:
Department of Veterinary Sciences and Public Health, University of Milan, Via Celoria 10, 20133 Milano, Italy
Gustavo G M Snel
Affiliation:
Department of Veterinary Sciences and Public Health, University of Milan, Via Celoria 10, 20133 Milano, Italy
Michela Malvisi
Affiliation:
Department of Veterinary Sciences and Public Health, University of Milan, Via Celoria 10, 20133 Milano, Italy
Renata Piccinini*
Affiliation:
Department of Veterinary Sciences and Public Health, University of Milan, Via Celoria 10, 20133 Milano, Italy
*
*For correspondence; e-mail: [email protected]
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Abstract

Staphylococcus aureus isolates from dairy cow mastitis are not always consistent with the characteristic morphology described, and molecular investigation is often needed. The aim of the study was to develop a duplex real-time PCR assay for rapid identification of Staph. aureus isolates, targeting both nuc and Sa442. Overall, 140 isolates collected from dairy cow mastitis in 90 different herds, were tested. All strains had been identified using morphological and biochemical characteristics. DNA from each strain was amplified in real-time PCR assay, to detect nuc or Sa442. Thereafter, a duplex real-time PCR assay was performed, and specificity of the amplified products was assessed by high resolution melting curve analysis. Out of 124 Staph. aureus isolates, 33 did not show the typical morphology or enzymic activity; in 118 strains, the two melt-curve peaks consistent with nuc and Sa442 were revealed, while 2 isolates showed only the peak consistent with Sa442. Four isolates bacteriologically identified as Staph. aureus, were PCR-negative and were further identified as Staph. pseudintermedius by sequencing. Staph. pseudintermedius and coagulase-negative staphylococci did not carry nuc or Sa442. The results showed the correct identification of all isolates, comprehending also coagulase—or nuc-negative Staph. aureus, while other coagulase-positive Staphylococci were correctly identified as non-Staph. aureus. Both sensitivity and specificity were 100%. High resolution melting analysis allowed easy detection of unspecific products. Finally, the duplex real-time PCR was applied directly to 40 milk samples, to detect infected mammary quarters. The assay confirmed the results of bacteriological analysis, on Staph. aureus-positive or—negative samples. Therefore, the proposed duplex real-time PCR could be used in laboratory routine as a cost-effective and powerful tool for high-throughput identification of atypical Staph. aureus isolates causing dairy cow mastitis. Also, it could be applied directly to milk samples, to detect Staph. aureus mammary infections avoiding bacteriological analysis.

Keywords

Staphylococcus aureusmastitisduplex real-time PCRdairy cow
Type
Research Article
Information
Journal of Dairy Research , Volume 80 , Issue 2 , May 2013 , pp. 223 - 226
Copyright
Copyright © Proprietors of Journal of Dairy Research 2013

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