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A PCR-Based Method for the Detectionof Streptococcus agalactiae in Milk

I. Meiri-Bendek^*, E. Lipkin, A. Friedmann, G. Leitner, A. Saran, S. Friedman and Y. Kashi^*

^* Faculty of Food Engineering and Biotechnology, Technion, 3200 Haifa, Israel
Dept. of Genetics, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel
National Mastitis Reference Center, Kimron Veterinary Institute, 50250 Bet Dagan, Israel
National Service for Udder Health and Milk Quality, Israel Cattle Breeders Association, 38900 Caesaria, Israel

Corresponding author:
Y. Kashi; e-mail:
kashi{at}tx.technion.ac.il.

Bovine mastitis caused by Streptococcus agalactiae is mainly subclinical and therefore can be diagnosed only in the laboratory. We developed a polymerase chain reaction (PCR)-based method for specific and sensitive detection of S. agalactiae in raw milk. The specificity of the PCR reaction is based on unique S. agalactiae DNA sequences within the 16S subunit of the rRNA genes. Two pairs of sequences were used as positive controls; general streptococci primers, which anneal to conserved areas within the 16S rRNA subunit gene, and primers, which anneal to sequences within bovine mitochondrial DNA. The method of detection includes selective enrichment of S. agalactiae in the milk sample, followed by DNA extraction using a rapid and simple procedure developed for this purpose, and specific PCR reaction with appropriate controls. The method enables the detection of one bacterium in 1 ml of raw milk. The method developed can be easily incorporated as part of routine screening of bulk milk collection tanks for early detection of infected cows in a herd.

Abbreviation key: CAMP = Christie, Atkins and Munch-Peterson

Key Words: Streptococcus agalactiae • bovine mastitis • polymerase chain reaction diagnosis

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