Quantitative detection of Escherichia coli from urine of patients with bacteriuria by real-time PCR

2004 ◽  
Vol 8 (3) ◽  
pp. 179-184 ◽  
Author(s):  
Nobuyuki Hinata ◽  
Toshiro Shirakawa ◽  
Hiroshi Okada ◽  
Katsumi Shigemura ◽  
Sadao Kamidono ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Detection

scholarly journals Evaluation of real-time PCR for quantitative detection of Escherichia coli in beach water

2013 ◽  
Vol 12 (1) ◽  
pp. 51-56 ◽  
Author(s):  
Jason Tszhin Lam ◽  
Edwin Lui ◽  
Simon Chau ◽  
Cathie Show Wu Kueh ◽  
Ying-kit Yung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Limit Of Quantification ◽  
E Coli ◽  
Polymerase Chain ◽  
Beach Water

The current investigation evaluated the use of real-time polymerase chain reaction (PCR) for quantitative detection of Escherichia coli in marine beach water. Densities of E. coli in 263 beach water samples collected from 13 bathing beaches in Hong Kong between November 2008 and December 2009 were determined using both real-time PCR and culture-based methods. Regression analysis showed that these two methods had a significant positive linear relationship with a correlation coefficient (r) of 0.64. Serial dilution of spiked samples indicated that the real-time PCR had a limit of quantification of 25 E. coli colonies in 100 mL water sample. This study showed that the rapid real-time PCR has potential to complement the traditional culture method of assessing fecal pollution in marine beach water.

Rapid and Simultaneous Quantitation of Escherichia coli O157:H7, Salmonella, and Shigella in Ground Beef by Multiplex Real-Time PCR and Immunomagnetic Separation†

2007 ◽  
Vol 70 (6) ◽  
pp. 1366-1372 ◽  
Author(s):  
LUXIN WANG ◽  
YONG LI ◽  
AZLIN MUSTAPHA
Keyword(s):  
Escherichia Coli ◽  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Quantitative Detection ◽  
Escherichia Coli O157 ◽  
The Real ◽  
E Coli

The objective of this study was to establish a multiplex real-time PCR for the simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella. Genomic DNA for the real-time PCR was extracted by the boiling method. Three sets of primers and corresponding TaqMan probes were designed to target these three pathogenic bacteria. Multiplex real-time PCR was performed with TaqMan Universal PCR Master Mix in an ABI Prism 7700 Sequence Detection System. Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log CFU per milliliter) via linear regression. With optimized conditions, the quantitative detection range of the real-time multiplex PCR for pure cultures was 102 to 109 CFU/ml for E. coli O157:H7, 103 to 109 CFU/ml for Salmonella, and 101 to 108 CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 105 CFU/g for E. coli O157:H7, 103 CFU/g for Salmonella, and 104 CFU/g for Shigella. Immunomagnetic separation (IMS) was further used to separate E. coli O157:H7 and Salmonella from the beef samples. With the additional use of IMS, the detection limit was 103 CFU/g for both pathogens. Results from this study showed that TaqMan real-time PCR, combined with IMS, is potentially an effective method for the rapid and reliable quantitation of E. coli O157:H7, Salmonella, and Shigella in food.

scholarly journals Association of Ct Values from Real-Time PCR with Culture in Microbiological Clearance Samples for Shiga Toxin-Producing Escherichia coli (STEC)

2020 ◽  
Vol 8 (11) ◽  
pp. 1801
Author(s):  
Michael Bording-Jorgensen ◽  
Brendon D. Parsons ◽  
Gillian A.M. Tarr ◽  
Binal Shah-Gandhi ◽  
Colin Lloyd ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Gene Detection ◽  
Culture Positivity ◽  
Hands On ◽  
Culture Independent ◽  
Chromogenic Agar ◽  
Stool Samples

Shiga toxin-producing Escherichia coli (STEC) are associated with acute gastroenteritis worldwide, which induces a high economic burden on both healthcare and individuals. Culture-independent diagnostic tests (CIDT) in frontline microbiology laboratories have been implemented in Alberta since 2019. The objectives of this study were to determine the association between gene detection and culture positivity over time using STEC microbiological clearance samples and also to establish the frequency of specimen submission. Both stx genes’ amplification by real-time PCR was performed with DNA extracted from stool samples using the easyMAG system. Stools were inoculated onto chromogenic agar for culture. An association between gene detection and culture positivity was found to be independent of which stx gene was present. CIDT can provide rapid reporting with less hands-on time and technical expertise. However, culture is still important for surveillance and early cluster detection. In addition, stool submissions could be reduced from daily to every 3–5 days until a sample is negative by culture.

Real-Time PCR Determination of Escherichia coli Genomic DNA Contamination in Plasmid Preparations

2002 ◽  
Vol 301 (1) ◽  
pp. 151-153 ◽  
Author(s):  
Adrián Vilalta ◽  
Vanessa Whitlow ◽  
Terrie Martin
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Dna Contamination

scholarly journals Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

2005 ◽  
Vol 71 (7) ◽  
pp. 3911-3916 ◽  
Author(s):  
Mark G. Wise ◽  
Gregory R. Siragusa
Keyword(s):  
Gastrointestinal Tract ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Quantitative Detection ◽  
Necrotic Enteritis ◽  
Analytical Sensitivity ◽  
Quantitative Real Time Pcr ◽  
Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

Real-time PCR for differentiation of F18 variants among enterotoxigenic and Shiga toxin-producing Escherichia coli from piglets with diarrhoea and oedema disease

2013 ◽  
Vol 198 (2) ◽  
pp. 538-540 ◽  
Author(s):  
Jae-Won Byun ◽  
Byeong Yeal Jung ◽  
Ha-Young Kim ◽  
John M. Fairbrother ◽  
Myoung-Heon Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Oedema Disease

scholarly journals Application of quantitative real-time PCR compared to filtration methods for the enumeration of Escherichia coli in surface waters within Vietnam

2016 ◽  
Vol 15 (1) ◽  
pp. 155-162 ◽  
Author(s):  
Pierangeli G. Vital ◽  
Nguyen Thi Van Ha ◽  
Le Thi Hong Tuyet ◽  
Kenneth W. Widmer
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Surface Waters ◽  
Real Time Pcr ◽  
Membrane Filtration ◽  
Quantitative Real Time Pcr ◽  
Wet Season ◽  
Culture Methods ◽  
Fecal Indicator ◽  
Filtration Method

Surface water samples in Vietnam were collected from the Saigon River, rural and suburban canals, and urban runoff canals in Ho Chi Minh City, Vietnam, and were processed to enumerate Escherichia coli. Quantification was done through membrane filtration and quantitative real-time polymerase chain reaction (PCR). Mean log colony-forming unit (CFU)/100 ml E. coli counts in the dry season for river/suburban canals and urban canals were log 2.8 and 3.7, respectively, using a membrane filtration method, while using Taqman quantitative real-time PCR they were log 2.4 and 2.8 for river/suburban canals and urban canals, respectively. For the wet season, data determined by the membrane filtration method in river/suburban canals and urban canals samples had mean counts of log 3.7 and 4.1, respectively. While mean log CFU/100 ml counts in the wet season using quantitative PCR were log 3 and 2, respectively. Additionally, the urban canal samples were significantly lower than those determined by conventional culture methods for the wet season. These results show that while quantitative real-time PCR can be used to determine levels of fecal indicator bacteria in surface waters, there are some limitations to its application and it may be impacted by sources of runoff based on surveyed samples.

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