scholarly journals Comparison of a Miniaturized Cassette PCR System with a Commercially Available Platform for Detecting Escherichia coli in Beef Carcass Swabs

Micromachines ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 959
Author(s):  
Dammika P. Manage ◽  
Jana Lauzon ◽  
Linda M. Pilarski ◽  
Patrick M. Pilarski ◽  
Lynn M. McMullen
Keyword(s):  
Escherichia Coli ◽  
Curve Analysis ◽  
Detection Sensitivity ◽  
Conventional Pcr ◽  
False Negatives ◽  
Melt Curve Analysis ◽  
E Coli ◽  
Highly Sensitive ◽  
Multiple Samples ◽  
Beef Carcass

Detection sensitivity of cassette PCR was compared with a commercial BAX® PCR system for detection of eae and stx genes in Escherichia coli from 806 beef carcass swabs. Cassette PCR detects multiple genetic markers on multiple samples using PCR and melt curve analysis. Conventional PCR served as a gold standard. Overall, for positive and negative concordance, cassette PCR was 98.6% concordant with conventional PCR, and BAX PCR was 65.4% concordant. Of 806 beef carcass swabs, 339 by cassette PCR and 84 by BAX PCR harbored eae + stx+E. coli. For BAX PCR reactions, 84% of eae+ swabs, 79% of stx+ swabs, and 86% of eae + stx+ swabs were also detected by cassette PCR. For cassette PCR reactions, 457 swabs were eae+ with only 117 scored as eae+ using BAX PCR for 26% positive concordance. For stx primers, cassette PCR scored 480 samples as stx+ but only 215 samples were stx+ by BAX PCR, giving 45% positive concordance. Importantly, cassette PCR scored 339 swabs as harboring eae + stx+ E. coli, but BAX PCR detected only 71 positives giving only 21% positive concordance, with many false negatives. Cassette PCR is a highly sensitive method for detection of STEC genes in E. coli found in carcass swabs.

An Aggregation-Induced Emission Material Labeling Antigen-Based Lateral Flow Immunoassay Strip for Rapid Detection of Escherichia coli O157:H7

2021 ◽  
pp. 247263032098193
Author(s):  
Cheng Liu ◽  
Shuiqin Fang ◽  
Yachen Tian ◽  
Youxue Wu ◽  
Meijiao Wu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rapid Detection ◽  
Foodborne Pathogen ◽  
Test Strip ◽  
Lateral Flow ◽  
Detection Sensitivity ◽  
Lateral Flow Immunoassay ◽  
Escherichia Coli O157 ◽  
Aggregation Induced Emission ◽  
E Coli

Escherichia coli O157:H7 ( E. coli O157:H7) is a dangerous foodborne pathogen, mainly found in beef, milk, fruits, and their products, causing harm to human health or even death. Therefore, the detection of E. coli O157:H7 in food is particularly important. In this paper, we report a lateral flow immunoassay strip (LFIS) based on aggregation-induced emission (AIE) material labeling antigen as a fluorescent probe for the rapid detection of E. coli O157:H7. The detection sensitivity of the strip is 105 CFU/mL, which is 10 times higher than that of the colloidal gold test strip. This method has good specificity and stability and can be used to detect about 250 CFU of E. coli O157:H7 successfully in 25 g or 25 mL of beef, jelly, and milk. AIE-LFIS might be valuable in monitoring food pathogens for rapid detection.

scholarly journals Quantifying within- and between-animal variation and uncertainty associated with counts of Escherichia coli O157 occurring in naturally infected cattle faeces

2008 ◽  
Vol 6 (31) ◽  
pp. 169-177 ◽  
Author(s):  
S.E Robinson ◽  
P.E Brown ◽  
E.J Wright ◽  
C.A Hart ◽  
N.P French
Keyword(s):  
Escherichia Coli ◽  
Statistical Model ◽  
Human Infection ◽  
Microbial Count ◽  
Infected Cattle ◽  
Explanatory Variables ◽  
E Coli ◽  
Fixed And Random Effects ◽  
Cattle Faeces ◽  
Multiple Samples

Cattle faeces are considered the most important reservoir for human infection with Escherichia coli O157. We have previously described shedding of E. coli O157 in the faeces of naturally infected cattle cohorts. However, the data require further investigation to quantify the uncertainty and variability in the estimates previously presented. This paper proposes a method for analysing both the presence and the quantity of E. coli O157 in cattle faecal samples, using two isolation procedures, one of which enumerates E. coli O157. The combination of these two measurements, which are fundamentally different in nature and yet measuring a common outcome, has necessitated the development of a novel statistical model for ascertaining the contribution of the various components of variation (both natural and observation induced) and for judging the influence of explanatory variables. Most of the variation within the sampling hierarchy was attributable to multiple samples from the same animal. The contribution of laboratory-level variation was found to be low. After adjusting for fixed and random effects, short periods of increased intensity of shedding were identified in individual animals. We conclude that within-animal variation is greater than between animals over time, and studies aiming to elucidate the dynamics of shedding should focus resources, sampling more within than between animals. These findings have implications for the identification of persistent high shedders and for assessing their role in the epidemiology of E. coli O157 in cattle populations. The development of this non-standard statistical model may have many applications to other microbial count data.

Characterization of novel ybjG and dacC variants in Escherichia coli

2013 ◽  
Vol 62 (11) ◽  
pp. 1728-1734 ◽  
Author(s):  
Dongguo Wang ◽  
Enping Hu ◽  
Jiayu Chen ◽  
Xiulin Tao ◽  
Katelyn Gutierrez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Multiple Drug Resistance ◽  
Multiple Drug ◽  
The Novel ◽  
Conventional Pcr ◽  
E Coli ◽  
Novel Variants ◽  
Restriction Sites ◽  
Genetic Structures

A total of 69 strains of Escherichia coli from patients in the Taizhou Municipal Hospital, China, were isolated, and 11 strains were identified that were resistant to bacitracin, chloramphenicol, tetracycline and erythromycin. These strains were PCR positive for at least two out of three genes, ybjG, dacC and mdfA, by gene mapping with conventional PCR detection. Conjugation experiments demonstrated that these genes existed in plasmids that conferred resistance. Novel ybjG and dacC variants were isolated from E. coli strains EC2163 and EC2347, which were obtained from the sputum of intensive care unit patients. Genetic mapping showed that the genes were located on 8200 kb plasmid regions flanked by EcoRI restriction sites. Three distinct genetic structures were identified among the 11 PCR-positive strains of E. coli, and two contained the novel ybjG and dacC variants. The putative amino acid differences in the ybjG and dacC gene variants were characterized. These results provide evidence for novel variants of ybjG and dacC, and suggest that multiple drug resistance in hospital strains of E. coli depends on the synergistic function of ybjG, dacC and mdfA within three distinct genetic structures in conjugative plasmids.

Potential pitfalls in the quantitative molecular detection ofEscherichia coliO157:H7 in environmental matrices

2006 ◽  
Vol 52 (5) ◽  
pp. 482-488 ◽  
Author(s):  
Rebekka R.E Artz ◽  
Lisa M Avery ◽  
Davey L Jones ◽  
Ken Killham
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Detection Sensitivity ◽  
Escherichia Coli O157 ◽  
Soil System ◽  
E Coli ◽  
Animal Wastes ◽  
Pcr Assays

The detection sensitivity and potential interference factors of a commonly used assay based on real-time polymerase chain reaction (PCR) for Escherichia coli O157:H7 using eae gene-specific primers were assessed. Animal wastes and soil samples were spiked with known replicate quantities of a nontoxigenic strain of E. coli O157:H7 in a viable or dead state and as unprotected DNA. The detection sensitivity and accuracy of real-time PCR for E. coli O157:H7 in animal wastes and soil is low compared to enrichment culturing. Nonviable cells and unprotected DNA were shown to produce positive results in several of the environmental samples tested, leading to potential overestimates of cell numbers due to prolonged detection of nonviable cells. This demonstrates the necessity for the specific calibration of real-time PCR assays in environmental samples. The accuracy of the eae gene–based detection method was further evaluated over time in a soil system against an activity measurement, using the bioluminescent properties of an E. coli O157:H7 Tn5luxCDABE construct. The detection of significant numbers of viable but nonculturable (VBNC) as well as nonviable and possibly physically protected cells as shown over a period of 90 days further complicates the use of real-time PCR assays for quick diagnostics in environmental samples and infers that enrichment culturing is still required for the final verification of samples found positive by real-time PCR methods.Key words: Escherichia coli O157:H7, real-time PCR, animal waste, soil, VBNC.

Microbiological Effects of Hand Washing at a Beef Carcass–Breaking Facility†

2003 ◽  
Vol 66 (3) ◽  
pp. 493-496 ◽  
Author(s):  
C. O. GILL ◽  
J. C. McGINNIS
Keyword(s):  
Escherichia Coli ◽  
Hand Washing ◽  
E Coli ◽  
Before And After ◽  
Disinfectant Solution ◽  
The Mean ◽  
Beef Carcass

The hands of workers in the carcass-breaking facility at a beef packing plant were sampled by rinsing. Total aerobes, coliforms, and Escherichia coli were enumerated for each sample. The numbers of bacteria recovered from duplicate groups of 25 hand samples collected before and after hands were washed with an antibacterial gel, rinsed in a disinfectant solution, washed with the gel and rinsed with the disinfectant, or washed in the disinfectant for 20 s were similar for samples collected before work began after breaks. The numbers of bacteria recovered from samples collected before and after hands were washed with the antibacterial gel and rinsed in the disinfectant solution were similar for samples collected after work as well. However, the mean numbers of aerobes recovered from the four groups of hand samples after work were all >6.5 log CFU per hand, while 9 of the 10 corresponding values for groups of hand samples collected before work were <6.5 log CFU per hand; the total numbers of coliforms recovered from three groups of hand samples collected after work were >4 log CFU/25 hands, while 9 of the corresponding values for groups of hand samples collected before work were <4 log CFU/25 hands. The total numbers of E. coli recovered from all groups of hand samples collected after work were >3.5 log CFU/25 hands, while 9 of the corresponding values for groups of hand samples collected before work were <3 log CFU/25 hands. Thus, although washing and/or rinsing apparently did not reduce the numbers of bacteria on hands, fewer bacteria were recovered from hands before than after work.

scholarly journals Characterization and Antibiogram of Escherichia coli Associated with Mortality in Broilers and Ducklings in Bangladesh

1970 ◽  
Vol 2 (1) ◽  
pp. 9-14 ◽  
Author(s):  
MT Islam ◽  
MA Islam ◽  
MA Samad ◽  
SML Kabir
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Sensitivity ◽  
High Rate ◽  
E Coli ◽  
Sensitivity Pattern ◽  
Highly Sensitive ◽  
Biochemical Studies ◽  
Increasing Trend ◽  
Escherichia Coli Infection ◽  
Hemorrhagic Enteritis

Mortality in broilers (6.56%) and growing ducks (11.0%) caused by Escherichia coli was recorded in the experimental flocks study during the period from May to August 2003. E. coli organisms isolated from broiler birds affected with characteristic lesions of omphalitis and yolk sac infection, fibrinous pericarditis and peri-hepatitis , hemorrhagic enteritis, and accumulation of excessive pericardial and peritoneal fluid, whereas from ducks with lesions of hemorrhagic enteritis and extensive epicardial hemorrhages. Each of the 21 isolates collected from broilers and 11 isolates from ducks was characterized by cultural and biochemical studies, of which 8 isolates from broilers and 5 isolates from ducks were tested for antibiotic sensitivity with 9 different antibiotics. The antibiotic sensitivity pattern showed that the isolates were highly sensitive to ciprofloxacin but an increasing trend of resistance was recorded in broilers (7 / 9) than duck (4 / 9) isolates. It may be concluded from the results of this study that the high rate of E. coli infection in broilers and ducks along with the high resistance of isolates to antibiotics constitute a threat to the poultry industry in Bangladesh. Key words: Escherichia coli infection; mortality; broilers; ducks; characterization; antibiogram doi: 10.3329/bjvm.v2i1.1927 Bangl. J. Vet. Med. (2004). 2 (1) : 09-14

Assessment of the Hygienic Performance of a Sheep Carcass Dressing Process†

1998 ◽  
Vol 61 (3) ◽  
pp. 329-333 ◽  
Author(s):  
C.O. GILL ◽  
L.P. BAKER
Keyword(s):  
Escherichia Coli ◽  
Standard Deviation ◽  
Arithmetic Mean ◽  
Single Sample ◽  
E Coli ◽  
The Mean ◽  
Beef Carcass ◽  
Normally Distributed

Swab samples were obtained from the surfaces of randomly selected carcasses passing through a sheep carcass-dressing process. A single sample was obtained from a randomly selected site on the surface of each carcass. Twenty-five such samples were collected at each of four stages in the process. The aerobio bacteria, coliforms, and Escherichia coli recovered from each sample were enumerated. Values for the mean log and standard deviation of each set of 25 log10 values were calculated on the assumption that the log values were normally distributed. The log of the arithmetic mean was estimated from the mean log and standard deviation values for each set. The results showed that bacteria, including coliforms that were largely E. coli, were deposited in high numbers during skinning operations, mainly on the butts and shoulders of carcasses. The mean numbers of coliforms and E. coli on carcasses were little affected by eviscerating and trimming operations, although they were redistributed from the sites they occupied after skinning. Total counts were redistributed and augmented by eviscerating and trimming operations. Washing reduced the log numbers of all of the bacteria by approximately 0.5. The general hygienic characteristics of the sheep carcass dressing process were similar to those of a previously examined beef carcass-dressing process.

Contamination of Beef Chucks with Escherichia coli during Carcass Breaking†

2001 ◽  
Vol 64 (11) ◽  
pp. 1824-1827 ◽  
Author(s):  
C. O. GILL ◽  
J. C. McGINNIS ◽  
J. BRYANT
Keyword(s):  
Escherichia Coli ◽  
Outer Surface ◽  
Conveyor Belt ◽  
E Coli ◽  
Beef Carcass

Samples were obtained by swabbing the whole of the chuck portion on each of the first 500 sides that entered a beef carcass breaking process and the whole of the outer surface of each of the chuck primal cuts that were prepared from those portions. Swabs obtained from groups of 10 sides or cuts that entered or emerged from the process consecutively were combined, and the coliforms and Escherichia coli recovered from each group were enumerated. Coliforms and E. coli were recovered only sporadically from groups of sides at log total numbers of 4.0 and 3.5 log CFU/500 sides, respectively. Coliforms were recovered from three and E. coli from none of the first six groups of cuts. Coliforms and E. coli were recovered from all subsequent groups of cuts, initially at log numbers mostly <3 log CFU/10 cuts, but ultimately at log numbers mostly >3 log CFU/10 cuts. The log total numbers of coliforms and E. coli recovered from cuts were >6.0 and 5.5 log CFU/500 cuts, respectively. After the breaking of about 600 sides, samples were obtained by swabbing a table onto which the part of the side that included the chuck portion was deposited after it was cut from the hanging side, and the belt that was used for conveying chucks. The numbers of coliforms and E. coli recovered from the table and conveyor belt were comparable with the numbers recovered from sides and cuts, respectively. Those findings show that most of the coliforms and E. coli recovered from the cuts were not present on carcass sides but that they originated largely from the cut conveying equipment.

Primers with 5′ Flaps Improve the Efficiency and Sensitivity of Multiplex PCR Assays for the Detection of Salmonella and Escherichia coli O157:H7

2013 ◽  
Vol 76 (4) ◽  
pp. 668-673 ◽  
Author(s):  
CHRIS TIMMONS ◽  
SHEFALI DOBHAL ◽  
JACQUELINE FLETCHER ◽  
LI MARIA MA
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Fold Increase ◽  
Detection Sensitivity ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
Robust Detection ◽  
E Coli ◽  
Pcr Multiplex ◽  
Pcr Assays

Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5′ end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5′ flap (5′-AATAAATCATAA-3′). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection.

Comparing Real-Time and Conventional PCR to Culture-Based Methods for Detecting and Quantifying Escherichia coli O157 in Cattle Feces

2014 ◽  
Vol 77 (2) ◽  
pp. 314-319 ◽  
Author(s):  
M. E. JACOB ◽  
J. BAI ◽  
D. G. RENTER ◽  
A. T. ROGERS ◽  
X. SHI ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Positive Culture ◽  
Feedlot Cattle ◽  
Escherichia Coli O157 ◽  
Conventional Pcr ◽  
Culture Methods ◽  
E Coli ◽  
Cattle Feces ◽  
Culture Negative

Detection of Escherichia coli O157 in cattle feces has traditionally used culture-based methods; PCR-based methods have been suggested as an alternative. We aimed to determine if multiplex real-time (mq) or conventional PCR methods could reliably detect cattle naturally shedding high (≥104 CFU/g of feces) and low (~102 CFU/g of feces) concentrations of E. coli O157. Feces were collected from pens of feedlot cattle and evaluated for E. coli O157 by culture methods. Samples were categorized as (i) high shedders, (ii) immunomagnetic separation (IMS) positive after enrichment, or (iii) culture negative. DNA was extracted pre- and postenrichment from 100 fecal samples from each category (high shedder, IMS positive, culture negative) and subjected to mqPCR and conventional PCR assays based on detecting three genes, rfbE, stx1, and stx2. In feces from cattle determined to be E. coli O157 high shedders by culture, 37% were positive by mqPCR prior to enrichment; 85% of samples were positive after enrichment. In IMS-positive samples, 4% were positive by mqPCR prior to enrichment, while 43% were positive after enrichment. In culture-negative feces, 7% were positive by mqPCR prior to enrichment, and 40% were positive after enrichment. The proportion of high shedder–positive and culture-positive (high shedder and IMS) samples were significantly different from mqPCR-positive samples before and after enrichment (P < 0.01). Similar results were observed for conventional PCR. Our data suggest that mqPCR and conventional PCR are most useful in identifying high shedder animals and may not be an appropriate substitute to culture-based methods for detection of E. coli O157 in cattle feces.

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