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Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens

(2009) JOURNAL OF DAIRY SCIENCE. 92(3). p.952-959
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Organization
Abstract
Intramammary infection (IMI), also known as mastitis, is the most frequently occurring and economically the most important infectious disease in dairy cattle. This study provides a validation of the analytical specificity and sensitivity of a real-time PCR-based assay that identifies 11 major pathogen species or species groups responsible for IMI, and a gene coding for staphylococcal beta-lactamase production (penicillin resistance). Altogether, 643 culture isolates originating from clinical bovine mastitis, human, and companion animal samples were analyzed using the assay. The isolates represented 83 different species, groups, or families, and originated from 6 countries in Europe and North America. The analytical specificity and sensitivity of the assay was 100% in bacterial and beta-lactamase identification across all isolates originating from bovine mastitis (n = 454). When considering the entire culture collection (including also the isolates originating from human and companion animal samples), 4 Streptococcus pyogenes, 1 Streptococcus salivarius, and 1 Streptococcus sanguis strain of human origin were identified as Streptococcus uberis, and 3 Shigella spp. strains were identified as Escherichia coli, decreasing specificity to 99% in Strep. uberis and to 99.5% in E. coli. These false-positive results were confirmed by sequencing of the 16S rRNA gene. Specificity and sensitivity remained at 100% for all other bacterial targets across the entire culture collection. In conclusion, the real-time PCR assay shows excellent analytical accuracy and holds much promise for use in routine bovine IMI testing programs. This study provides the basis for evaluating the assay's diagnostic performance against the conventional bacterial culture method in clinical field trials using mastitis milk samples.
Keywords
real-time PCR, bovine intramammary infection, validation, STAPHYLOCOCCUS-AUREUS, MILK SAMPLES, STREPTOCOCCUS-AGALACTIAE, SCREENING-TEST, PREVALENCE, UBERIS, TOOL, PCR

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Citation

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MLA
Koskinen, MT et al. “Analytical Specificity and Sensitivity of a Real-time Polymerase Chain Reaction Assay for Identification of Bovine Mastitis Pathogens.” JOURNAL OF DAIRY SCIENCE 92.3 (2009): 952–959. Print.
APA
Koskinen, M., Holopainen, J., Pyorala, S., Bredbacka, P., Pitkala, A., Barkema, H., Bexiga, R., et al. (2009). Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens. JOURNAL OF DAIRY SCIENCE, 92(3), 952–959.
Chicago author-date
Koskinen, MT, J Holopainen, S Pyorala, P Bredbacka, A Pitkala, Herman Barkema, R Bexiga, et al. 2009. “Analytical Specificity and Sensitivity of a Real-time Polymerase Chain Reaction Assay for Identification of Bovine Mastitis Pathogens.” Journal of Dairy Science 92 (3): 952–959.
Chicago author-date (all authors)
Koskinen, MT, J Holopainen, S Pyorala, P Bredbacka, A Pitkala, Herman Barkema, R Bexiga, J Roberson, L Solverod, R Piccinini, D Kelton, H Lehmusto, S Niskala, and L Salmikivi. 2009. “Analytical Specificity and Sensitivity of a Real-time Polymerase Chain Reaction Assay for Identification of Bovine Mastitis Pathogens.” Journal of Dairy Science 92 (3): 952–959.
Vancouver
1.
Koskinen M, Holopainen J, Pyorala S, Bredbacka P, Pitkala A, Barkema H, et al. Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens. JOURNAL OF DAIRY SCIENCE. 2009;92(3):952–9.
IEEE
[1]
M. Koskinen et al., “Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens,” JOURNAL OF DAIRY SCIENCE, vol. 92, no. 3, pp. 952–959, 2009.
@article{696116,
  abstract     = {Intramammary infection (IMI), also known as mastitis, is the most frequently occurring and economically the most important infectious disease in dairy cattle. This study provides a validation of the analytical specificity and sensitivity of a real-time PCR-based assay that identifies 11 major pathogen species or species groups responsible for IMI, and a gene coding for staphylococcal beta-lactamase production (penicillin resistance). Altogether, 643 culture isolates originating from clinical bovine mastitis, human, and companion animal samples were analyzed using the assay. The isolates represented 83 different species, groups, or families, and originated from 6 countries in Europe and North America. The analytical specificity and sensitivity of the assay was 100% in bacterial and beta-lactamase identification across all isolates originating from bovine mastitis (n = 454). When considering the entire culture collection (including also the isolates originating from human and companion animal samples), 4 Streptococcus pyogenes, 1 Streptococcus salivarius, and 1 Streptococcus sanguis strain of human origin were identified as Streptococcus uberis, and 3 Shigella spp. strains were identified as Escherichia coli, decreasing specificity to 99% in Strep. uberis and to 99.5% in E. coli. These false-positive results were confirmed by sequencing of the 16S rRNA gene. Specificity and sensitivity remained at 100% for all other bacterial targets across the entire culture collection. In conclusion, the real-time PCR assay shows excellent analytical accuracy and holds much promise for use in routine bovine IMI testing programs. This study provides the basis for evaluating the assay's diagnostic performance against the conventional bacterial culture method in clinical field trials using mastitis milk samples.},
  author       = {Koskinen, MT and Holopainen, J and Pyorala, S and Bredbacka, P and Pitkala, A and Barkema, Herman and Bexiga, R and Roberson, J and Solverod, L and Piccinini, R and Kelton, D and Lehmusto, H and Niskala, S and Salmikivi, L},
  issn         = {0022-0302},
  journal      = {JOURNAL OF DAIRY SCIENCE},
  keywords     = {real-time PCR,bovine intramammary infection,validation,STAPHYLOCOCCUS-AUREUS,MILK SAMPLES,STREPTOCOCCUS-AGALACTIAE,SCREENING-TEST,PREVALENCE,UBERIS,TOOL,PCR},
  language     = {eng},
  number       = {3},
  pages        = {952--959},
  title        = {Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens},
  url          = {http://dx.doi.org/10.3168/jds.2008-1549},
  volume       = {92},
  year         = {2009},
}

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