The specific and sensitive detection of foodborne pathogens in milk by PCR [Polymerase Chain Reaction]
1996
Schraft, H. | Laberge, I. | Griffiths, M.W. (Guelph Univ. (Canada). Dept. of Food Science)
Pathogens are often present in raw milk in low numbers which cannot be readily detected or enumerated by conventional cultural techniques. The use of the polymerase chain reaction in combination with a digoxigenin (DIG)-labeled internal hybridization probe allows the sensitive and specific detection of bacterial and protozoal contaminants of raw milk. PCR protocols have been designed for the amplification of a sequence of the cereolysin gene of Bacillus cereus and B. cereus-gp. species directly in milk. An internal hybridization probe labeled with DIG was used to hybridize with the ampfified product and the hybridization was detected with an anti-DIG antibody labeled with alkaline phosphatase. The reaction of the enzyme with a chemiluminescent substrate was monitored using a CCD photon counting camera. This technique allowed the detection of as low as 1 B. cereus cell in 20 ml of milk without the need for prior enrichment. A similar technique, based on PCR-amplification of an oocyst coat-protein gene, was used for the detection of Cryptosporidium parvum oocysts in raw milk. Again the procedure allowed reliable detection of 5 oocysts in 20 ml of milk and, on occasion, 1 oocyst could be detected.
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