Evaluation of a Multiplex Real-Time Polymerase Chain Reaction for the Quantification of Escherichia coli O157 in Cattle Feces

2012 ◽  
Vol 9 (1) ◽  
pp. 79-85 ◽  
Author(s):  
Megan E. Jacob ◽  
Xiaorong Shi ◽  
Baoyan An ◽  
Tiruvoor G. Nagaraja ◽  
Jianfa Bai
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
Cattle Feces ◽  
Polymerase Chain

A rapid (one day), sensitive real-time polymerase chain reaction assay for detecting Escherichia coli O157:H7 in ground beef

2006 ◽  
Vol 52 (10) ◽  
pp. 992-998 ◽  
Author(s):  
Jane Holicka ◽  
Rebecca A Guy ◽  
Anita Kapoor ◽  
David Shepherd ◽  
Paul A Horgen
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Polymerase Chain Reaction Assay ◽  
Ground Beef ◽  
Automatic Process ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain

The purpose of this study was to apply our rapid, integrated double enrichment 5′ nuclease real-time polymerase chain reaction assay for the detection of Escherichia coli O157:H7 and evaluate its efficacy. The assay targeted ground beef, an important vehicle in disease epidemiology. The assay reliably determined in 8 h the presence of E. coli O157:H7 in ground beef at the level of 1 colony-forming unit (CFU)/g. The sensitivity and specificity of the assay were compared with that of standard enrichment diagnostic techniques. A correlation of 100% in detection was achieved to the limit of 1 CFU/g. This assay can be used as a rapid, automatic process for identification of E. coli O157:H7 in ground beef or can be integrated with standard culture procedures, resulting in considerable cost and time savings.Key words: real-time PCR, E. coli O157:H7, ground beef, molecular diagnostics, rapid O157:H7 assay.

Polymerase Chain Reaction Assay for Detection of Escherichia coli O157:H7 and Escherichia coli O157:H−

2002 ◽  
Vol 65 (1) ◽  
pp. 5-11 ◽  
Author(s):  
TAKAHISA MIYAMOTO ◽  
NATSUKO ICHIOKA ◽  
CHIE SASAKI ◽  
HIROSHI KOBAYASHI ◽  
KEN-ICHI HONJOH ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Dna Sequence ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
Pcr Assay ◽  
E Coli ◽  
Pcr Product ◽  
Genomic Dnas ◽  
Polymerase Chain

The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non–E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H− and E. coli O157:H7. Since the DNA sequence from base 15 to base 1008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H− and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 × 100 to 3.5 × 102 CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42°C for 18 h and for the conventional cultural method.

Detection of the Escherichia coli pathogenic geneeaewith three real-time polymerase chain reaction methods

2007 ◽  
Vol 53 (3) ◽  
pp. 398-403 ◽  
Author(s):  
Joanne  Karen McCrea ◽  
Chenyi Liu ◽  
Lai-King Ng ◽  
Gehua Wang
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Detection Limit ◽  
Real Time ◽  
Turnaround Time ◽  
Sybr Green ◽  
Sybr Green I ◽  
Chain Reaction ◽  
Culture Techniques ◽  
Polymerase Chain

Several real-time polymerase chain reaction (PCR) methods are currently available to rapidly detect the presence of a specific DNA sequence. When used for detection of pathogenic organisms, the turnaround time for PCR-based methods is much lower than for traditional culture techniques. This study compared the sensitivity of three real-time PCR methods when detecting the Escherichia coli pathogenic gene eae to determine which method is most effective in identifying very low levels of the organism. The three methods were used to detect the eae gene over a range of DNA concentrations. The differences in sensitivity were statistically significant (p < 0.05), and SYBR Green I PCR was found to have the lowest detection limit of the three; LUX primers had the highest detection limit. Therefore, using a defined DNA concentration for detecting the eae gene, SYBR Green I is the best alternative.

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