Evaluation of a 5′-Nuclease (TaqMan) Assay for the Detection of Virulent Strains of Yersinia enterocolitica in Raw Meat and Tofu Samples†

2001 ◽  
Vol 64 (3) ◽  
pp. 355-360 ◽  
Author(s):  
A. VISHNUBHATLA ◽  
R. D. OBERST ◽  
D. Y. C. FUNG ◽  
W. WONGLUMSOM ◽  
M. P. HAYS ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Detection System ◽  
Detection Methods ◽  
Taqman Assay ◽  
Contamination Rate ◽  
Virulence Plasmid ◽  
Recovery Method ◽  
Ground Pork ◽  
Pure Cultures ◽  
Nuclease Assay

Culture methods for detecting virulent Yersinia enterocolitica require selective enrichment and a series of confirmatory tests that are time-consuming, costly, and laborious. The objective of this study was to evaluate a fluorogenic 5′-nuclease assay for detecting the enterotoxin yst gene of virulent Y. enterocolitica in pure cultures, inoculated ground pork samples, and naturally contaminated food samples. These results were then compared with “gold standard” methods recommended by the U.S. Food and Drug Administration in the Bacteriological Analytical Manual for detecting pathogenic Y. enterocolitica. The 5′-nuclease assay was able to identify the organism in 100% of the repetitions when 102 CFU/ml or more organisms were present in pure cultures and 103 CFU/g or more organisms were present in ground pork. Similar recovery efficiency on cefsulodin-irgasan-novobiocin (CIN) agar plates was only evident when 105 CFU/ml or more organisms were present in pure culture and 106 CFU/g or more organisms were present in inoculated ground pork. The 5′-nuclease assay indicated a contamination rate of 35.5% (94/265) in various meats and tofu, whereas the CIN plating method indicated a contamination rate of 28.3% (75/265). This resulted in 100% sensitivity and 64.5% specificity for the 5′-nuclease assay when compared with the standard culture recovery method. Only 75% (60/80) of the Yersinia spp. isolated on CIN was identified as containing a virulence plasmid by autoagglutination and crystal violet binding tests. These results indicate that the true rate of contamination of virulent Y. enterocolitica in pork and other processed meats and foods is being underestimated using current detection methods. This study demonstrates the potential of the 5′-nuclease assay for rapidly and specifically detecting virulent Y. enterocolitica in processed foods with the added advantage of being an automated detection system with high-throughput capability.

scholarly journals Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica

2000 ◽  
Vol 66 (9) ◽  
pp. 4131-4135 ◽  
Author(s):  
A. Vishnubhatla ◽  
D. Y. C. Fung ◽  
R. D. Oberst ◽  
M. P. Hays ◽  
T. G. Nagaraja ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Pcr Amplification ◽  
Enteric Bacteria ◽  
Taqman Assay ◽  
Virulent Strains ◽  
Field Samples ◽  
Ground Pork ◽  
Standard Culture ◽  
Pure Cultures ◽  
Nuclease Assay

ABSTRACT We have developed a rapid procedure for the detection of virulentYersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5′ nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species ofYersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri,Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia,Salmonella, Citrobacter, andFlavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be ≥102 CFU/ml in pure cultures and ≥103 CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked withY. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocoliticawith both 5′ nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.

Evaluation of a 5′ -Nuclease (TaqMan) Assay with the Thin Agar Layer Oxyrase Method for the Detection of Yersinia enterocolitica in Ground Pork Samples†

2004 ◽  
Vol 67 (2) ◽  
pp. 271-277 ◽  
Author(s):  
V. C. H. WU ◽  
D. Y. C. FUNG ◽  
R. D. OBERST
Keyword(s):  
Detection Limit ◽  
Yersinia Enterocolitica ◽  
Food Samples ◽  
Selective Medium ◽  
Taqman Assay ◽  
Ground Pork ◽  
Sensitivity Studies ◽  
Agar Layer ◽  
Pork Samples ◽  
Nuclease Assay

A 5′-nuclease (TaqMan) assay was evaluated for its capability to recover and detect stressed Yersinia enterocolitica. Sensitivity studies of a 5′-nuclease assay for detecting Y. enterocolitica O:8 in a pure culture system and spiked ground pork samples demonstrated that the assay has reliable sensitivity with a detection limit of 3 to 4 log CFU/ml or CFU/g. The PCR 5′-nuclease (TaqMan) assay was evaluated with the Thin Agar Layer Oxyrase method (TALO, overlaying 14 ml of Trypticase soy agar with a 1:30 dilution of “Oxyrase® for Agar” onto a prepoured pathogen-specific, selective medium), and it was compared against the selective medium cefsulodin-irgasan-novobiocin (CIN) for recovering and detecting Y. enterocolitica from inoculated nonfrozen and frozen (−15°C, 2 days) ground pork samples. The TALO method showed more sensitivity (detection limit, 2 log CFU/ml), and it has greater recovery capability (0.5 to 1 log CFU/ml) than CIN (P < 0.05). The 5′-nuclease assay provided rapid detection processing (5 versus 24 h after an 18-h enrichment). The sensitivity per PCR was calculated to as low as 0 to 1 log CFU per PCR reaction; however, in the method's current developmental stage, target pathogens should be enriched to 3 to 4 log CFU/ml or CFU/g to show consistent results. In a survey of 100 ground pork samples using TALO, CIN, and PCR methods, no Y. enterocolitica was recovered. A combined cultivation and an automated PCR TaqMan could be used as a presumptive screening test for detecting Y. enterocolitica in food samples.

Radiation Resistance of Virulence Plasmid-Containing and Plasmid-Less Yersinia enterocolitica†

2002 ◽  
Vol 65 (3) ◽  
pp. 556-559 ◽  
Author(s):  
CHRISTOPHER H. SOMMERS ◽  
JOHN S. NOVAK
Keyword(s):  
Radiation Dose ◽  
Ionizing Radiation ◽  
Yersinia Enterocolitica ◽  
Immune Suppression ◽  
Radiation Resistance ◽  
Type Iii Secretion ◽  
Foodborne Pathogen ◽  
Virulence Plasmid ◽  
Type Iii ◽  
Ground Pork

Yersinia enterocolitica, a foodborne pathogen, can be eliminated from meat by ionizing radiation. Y. enterocolitica sometimes contains a 70-kb virulence plasmid that encodes genes for a type III secretion channel and host immune suppression factors. The radiation resistance of virulence plasmid-containing and plasmid-less Y. enterocolitica was determined. Four Y. enterocolitica serotypes containing (i) the large virulence plasmid, and (ii) their plasmid-less derivatives were inoculated into raw ground pork, which was then vacuum packed and irradiated at 4°C to doses of 0.2, 0.4, 0.6, 0.8, and 1.0 kGy. The D10-value, the radiation dose required to reduce the number of viable Y. enterocolitica by 90%, was not dependent on the presence or absence of the virulence plasmid, but it did differ among the four Y. enterocolitica serotypes.

scholarly journals Rapid, Quantitative PCR Monitoring of Growth ofClostridium botulinum Type E in Modified-Atmosphere-Packaged Fish

2001 ◽  
Vol 67 (1) ◽  
pp. 206-216 ◽  
Author(s):  
B. Kimura ◽  
S. Kawasaki ◽  
H. Nakano ◽  
T. Fujii
Keyword(s):  
Quantitative Pcr ◽  
Detection System ◽  
Good Method ◽  
Taqman Assay ◽  
Toxin Gene ◽  
Modified Atmosphere ◽  
Sequence Detection ◽  
Guanidine Isothiocyanate ◽  
Fish Samples ◽  
Pure Cultures

ABSTRACT A rapid, quantitative PCR assay (TaqMan assay) which quantifiesClostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains ofC. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 102 to 108 CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.

PCR-Based DNA Amplification and Presumptive Detection of Escherichia coli O157:H7 with an Internal Fluorogenic Probe and the 5′ Nuclease (TaqMan) Assay

1998 ◽  
Vol 64 (9) ◽  
pp. 3389-3396 ◽  
Author(s):  
R. D. Oberst ◽  
M. P. Hays ◽  
L. K. Bohra ◽  
R. K. Phebus ◽  
C. T. Yamashiro ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Detection System ◽  
False Negative ◽  
Detection Threshold ◽  
Ground Beef ◽  
Taqman Assay ◽  
Dna Recovery ◽  
E Coli ◽  
Presumptive Identification ◽  
Nuclease Assay

ABSTRACT Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC)eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5′ nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5′ nuclease assay system was sufficient to correctly identify all E. coliO157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed,eaeA-targeted 5′ nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coliO157:H7 when ≥103 CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when ≥104 CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to ≥102 CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when ≥104 CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed,eaeA-targeted 5′ nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.

scholarly journals Adaptive Detection of Direct-Sequence Spread-Spectrum Signals Based on Knowledge-Enhanced Compressive Measurements and Artificial Neural Networks

Sensors ◽  
2021 ◽  
Vol 21 (7) ◽  
pp. 2538
Author(s):  
Shuang Zhang ◽  
Feng Liu ◽  
Yuang Huang ◽  
Xuedong Meng
Keyword(s):  
Neural Networks ◽  
Artificial Neural Networks ◽  
Spread Spectrum ◽  
High Efficiency ◽  
Detection System ◽  
Direct Sequence Spread Spectrum ◽  
Detection Performance ◽  
Detection Methods ◽  
Direct Sequence ◽  
Artificial Neural

The direct-sequence spread-spectrum (DSSS) technique has been widely used in wireless secure communications. In this technique, the baseband signal is spread over a wider bandwidth using pseudo-random sequences to avoid interference or interception. In this paper, the authors propose methods to adaptively detect the DSSS signals based on knowledge-enhanced compressive measurements and artificial neural networks. Compared with the conventional non-compressive detection system, the compressive detection framework can achieve a reasonable balance between detection performance and sampling hardware cost. In contrast to the existing compressive sampling techniques, the proposed methods are shown to enable adaptive measurement kernel design with high efficiency. Through the theoretical analysis and the simulation results, the proposed adaptive compressive detection methods are also demonstrated to provide significantly enhanced detection performance efficiently, compared to their counterpart with the conventional random measurement kernels.

scholarly journals A novel GPU based intrusion detection system using deep autoencoder with Fruitfly optimization

2021 ◽  
Vol 3 (6) ◽  
Author(s):  
R. Sekhar ◽  
K. Sasirekha ◽  
P. S. Raja ◽  
K. Thangavel
Keyword(s):  
Intrusion Detection ◽  
Intrusion Detection System ◽  
Missing Values ◽  
Detection System ◽  
Radial Basis Function Network ◽  
Detection Methods ◽  
Support Vector ◽  
Parameter Method ◽  
List Type ◽  
Fuzzy C Means

Abstract Intrusion Detection Systems (IDSs) have received more attention to safeguarding the vital information in a network system of an organization. Generally, the hackers are easily entering into a secured network through loopholes and smart attacks. In such situation, predicting attacks from normal packets is tedious, much challenging, time consuming and highly technical. As a result, different algorithms with varying learning and training capacity have been explored in the literature. However, the existing Intrusion Detection methods could not meet the desired performance requirements. Hence, this work proposes a new Intrusion Detection technique using Deep Autoencoder with Fruitfly Optimization. Initially, missing values in the dataset have been imputed with the Fuzzy C-Means Rough Parameter (FCMRP) algorithm which handles the imprecision in datasets with the exploit of fuzzy and rough sets while preserving crucial information. Then, robust features are extracted from Autoencoder with multiple hidden layers. Finally, the obtained features are fed to Back Propagation Neural Network (BPN) to classify the attacks. Furthermore, the neurons in the hidden layers of Deep Autoencoder are optimized with population based Fruitfly Optimization algorithm. Experiments have been conducted on NSL_KDD and UNSW-NB15 dataset. The computational results of the proposed intrusion detection system using deep autoencoder with BPN are compared with Naive Bayes, Support Vector Machine (SVM), Radial Basis Function Network (RBFN), BPN, and Autoencoder with Softmax. Article Highlights A hybridized model using Deep Autoencoder with Fruitfly Optimization is introduced to classify the attacks. Missing values have been imputed with the Fuzzy C-Means Rough Parameter method. The discriminate features are extracted using Deep Autoencoder with more hidden layers.

scholarly journals A Synthetic Biology Approach Using Engineered Bacteria to Detect Perfluoroalkyl Substance (PFAS) Contamination in Water

2021 ◽  
Vol 186 (Supplement_1) ◽  
pp. 801-807
Author(s):  
Nathaniel A Young ◽  
Ryan L Lambert ◽  
Angela M Buch ◽  
Christen L Dahl ◽  
Jackson D Harris ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Dna Sequences ◽  
Detection System ◽  
The United States ◽  
Detection Methods ◽  
Health Concern ◽  
Contaminated Water ◽  
Engineered Bacteria ◽  
Synthetic Biology Approach ◽  
Rhodococcus Jostii

ABSTRACT Introduction Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic compounds used industrially for a wide variety of applications. These PFAS compounds are very stable and persist in the environment. The PFAS contamination is a growing health issue as these compounds have been reported to impact human health and have been detected in both domestic and global water sources. Contaminated water found on military bases poses a potentially serious health concern for active duty military, their families, and the surrounding communities. Previous detection methods for PFAS in contaminated water samples require expensive and time-consuming testing protocols that limit the ability to detect this important global pollutant. The main objective of this work was to develop a novel detection system that utilizes a biological reporter and engineered bacteria as a way to rapidly and efficiently detect PFAS contamination. Materials and Methods The United States Air Force Academy International Genetically Engineered Machine team is genetically engineering Rhodococcus jostii strain RHA1 to contain novel DNA sequences composed of a propane 2-monooxygenase alpha (prmA) promoter and monomeric red fluorescent protein (mRFP). The prmA promoter is activated in the presence of PFAS and transcribes the mRFP reporter. Results The recombinant R. jostii containing the prmA promoter and mRFP reporter respond to exposure of PFAS by activating gene expression of the mRFP. At 100 µM of perfluorooctanoic acid, the mRFP expression was increased 3-fold (qRT-PCR). Rhodococcus jostii without exposure to PFAS compounds had no mRFP expression. Conclusions This novel detection system represents a synthetic biology approach to more efficiently detect PFAS in contaminated samples. With further refinement and modifications, a similar system could be readily deployed in the field around the world to detect this critical pollutant.

scholarly journals Identification and Characterization of Pathogenic Yersinia enterocolitica Isolates by PCR and Pulsed-Field Gel Electrophoresis

2005 ◽  
Vol 71 (7) ◽  
pp. 3674-3681 ◽  
Author(s):  
S. Thisted Lambertz ◽  
M.-L. Danielsson-Tham
Keyword(s):  
Multiplex Pcr ◽  
Yersinia Enterocolitica ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Virulence Plasmid ◽  
Pork Meat ◽  
Pulsed Field ◽  
Pork Products ◽  
Food Borne ◽  
Pork Samples

ABSTRACT Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n = 48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.

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