Rapid and sensitive immunomagnetic separation–polymerase chain reaction method for the detection of Escherichia coli O157[ratio ]H7 in raw milk and ice-cream

1997 ◽  
Vol 64 (1) ◽  
pp. 87-93 ◽  
Author(s):  
CHRISTOPHER M. GOODING ◽  
PRABHAKARA V. CHOUDARY
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Ice Cream ◽  
Raw Milk ◽  
Immunomagnetic Separation ◽  
Polymerase Chain Reaction Method ◽  
Escherichia Coli O157 ◽  
Agarose Gel Electrophoresis ◽  
Chain Reaction ◽  
Polymerase Chain

Escherichia coli O157[ratio ]H7 in spiked samples of raw milk and ice-cream was enriched in tryptic soy broth for 4 h, captured by immunomagnetic separation, subjected to amplification by polymerase chain reaction of parts of the verotoxin genes (SLT-I and SLT-II), and detected by agarose gel electrophoresis. Using this method, as few as 1 cfu Esch. coli O157[ratio ]H7/g food could be detected in <10 h.

Molecular Beacon Polymerase Chain Reaction Detection of Escherichia coli O157:H7 in Milk

2000 ◽  
Vol 63 (7) ◽  
pp. 855-859 ◽  
Author(s):  
JOHN L. McKILLIP ◽  
MARYANNE DRAKE
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Gel Electrophoresis ◽  
Molecular Beacon ◽  
Skim Milk ◽  
Agarose Gel ◽  
Escherichia Coli O157 ◽  
Agarose Gel Electrophoresis ◽  
Chain Reaction ◽  
Polymerase Chain

A fluorescently labeled oligonucleotide probe (molecular beacon) was applied to detect Escherichia coli O157:H7 in artificially contaminated skim milk during polymerase chain reaction (PCR) amplification of extracted DNA. The probe was designed to hybridize with a region of the slt-II gene coding for the A subunit and to fluoresce when the hairpin-stem conformation was linearized upon hybridization to the target sequence. The molecular beacon was incorporated into PCR reactions containing DNA extracted from artificially contaminated skim milk. The degree of fluorescence was monitored in PCR reactions containing 103, 105, and 107 CFU of E. coli O157:H7 per ml and was found to correlate with the amount of template in each reaction. Fluorescence significantly increased above background levels by cycle 8, 14, or 14 in reactions containing DNA from the 107-, 105-, or 103-CFU/ml template, respectively (P &lt; 0.05). Molecular beacon PCR demonstrated positive results more rapidly than traditional agarose gel electrophoresis analysis of PCR products. Use of molecular beacons allows real-time monitoring of PCR reactions, and the closed-tube format allows simultaneous detection and confirmation of target amplicons without the need for agarose gel electrophoresis and/or Southern blotting. This is the first report of a stem-and-loop molecular beacon being applied for direct detection of a pathogen in food.

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