Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction

被引:89
作者
Gillespie, BE [1 ]
Oliver, SP [1 ]
机构
[1] Univ Tennessee, Dept Anim Sci, Food Safety Ctr Excellence, Knoxville, TN 37996 USA
关键词
multiplex real-time polymerase chain reaction; Staphylococcus aureus; Streptococcus agalactiae; Streptococcus uberis;
D O I
10.3168/jds.S0022-0302(05)73036-8
中图分类号
S8 [畜牧、 动物医学、狩猎、蚕、蜂];
学科分类号
0905 ;
摘要
The objective of this study was to develop a multiplex real-time polymerase chain reaction (PCR) method for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk. A genetic marker specific for Staph. aureus was used for primers and dual-labeled probe design. The target for Strep. agalactiae primers and dual-labeled probe was selected from the cfb gene encoding the Christie-Atkins-Munch-Petersen factor. The plasminogen activator gene was the target for primers and dual-labeled probe design for Strep. uberis. Quarter milk samples (n = 192) were analyzed by the multiplex real-time PCR assay and conventional microbiological methods. An additional 57 quarter milk samples were analyzed in a separate real-time PCR assay for Strep. agalactiae only. Using an overnight enrichment step, the real-time PCR technique correctly identified 96.4% of all quarter milk samples; 91.7% of Staph. aureus, 98.2% of Strep. agalactiae, and 100% of Strep. uberis. Results of conventional microbiological methods were used to determine the sensitivity and specificity of the multiplex real-time PCR procedure. The sensitivity of the procedure to correctly identify Staph. aureus, Strep. agalactiae, and Strep. uberis directly from milk was 95.5%, and the specificity was 99.6%. Results of this study indicate that the multiplex real-time PCR procedure has the potential to be a valuable diagnostic technique for simultaneous identification of Staph. aureus, Strep. agalactiae, and Strep. uberis directly from quarter milk samples.
引用
收藏
页码:3510 / 3518
页数:9
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  • [21] The mechanism of a bacterial plasminogen activator intermediate between streptokinase and staphylokinase
    Sazonova, IY
    Houng, AK
    Chowdhry, SA
    Robinson, BR
    Hedstrom, L
    Reed, GL
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (16) : 12609 - 12613
  • [22] Inhibition of PCR in genital and urine specimens submitted for Chlamydia trachomatis testing
    Toye, B
    Woods, W
    Bobrowska, M
    Ramotar, K
    [J]. JOURNAL OF CLINICAL MICROBIOLOGY, 1998, 36 (08) : 2356 - 2358