Effect of Enrichment Medium on Real-Time Detection of Salmonella enterica from Lettuce and Tomato Enrichment Cultures

2010 ◽  
Vol 73 (6) ◽  
pp. 1047-1056 ◽  
Author(s):  
LISA GORSKI ◽  
ANITA S. LIANG
Keyword(s):  
Real Time ◽  
Pcr Primers ◽  
Immunomagnetic Separation ◽  
Separation Method ◽  
Qualitative Assessment ◽  
Detection Methods ◽  
Enrichment Cultures ◽  
Peptone Water ◽  
Immunological Technique ◽  
Plant Microbiota

Three enrichment broths commonly used for detection of Salmonella (buffered peptone water [BPW], tryptic soy broth [TSB], and universal preenrichment broth [UPB]) were compared for use in real-time SYBR Green PCR detection of Salmonella introduced into enrichment cultures made from store-bought lettuce and tomatoes. The produce served as a source of normal plant microbiota to measure how well DNA-based detection methods for Salmonella work in a suspension of plant-associated bacteria that may be closely related to Salmonella. A qualitative assessment of the background microbiota that grew in the three enrichment broths cultures from tomato and lettuce samples revealed that different bacteria predominated in the different broths. Results obtained with five produce-related outbreak Salmonella strains and PCR primers directed toward three different Salmonella genes suggest that the ability to detect Salmonella from these enrichment cultures by real-time PCR was 10 to 1,000 times better with TSB enrichment cultures. Detection levels were similar between the different enrichment media when an immunomagnetic separation method was used; however, the immunological technique did not enhance detection from TSB enrichment cultures. Detection could be affected by the medium and by the background microbiota. An immunomagnetic separation method may be useful in BPW and UPB enrichment cultures but not in TSB enrichment cultures.

scholarly journals Development of a Magnetic Capture Hybridization Real-Time PCR Assay for Detection of Tumorigenic Agrobacterium vitis in Grapevines

2013 ◽  
Vol 103 (6) ◽  
pp. 633-640 ◽  
Author(s):  
Kameka L. Johnson ◽  
Desen Zheng ◽  
Supaporn Kaewnum ◽  
Cheryl Lynn Reid ◽  
Thomas Burr
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Detection Threshold ◽  
Pcr Primers ◽  
Immunomagnetic Separation ◽  
Extraction Methods ◽  
Economic Effects ◽  
Agrobacterium Vitis ◽  
Magnetic Capture

Agrobacterium vitis, the causal agent of grape crown gall, can have severe economic effects on grape production. The bacterium survives systemically in vines and, therefore, is disseminated in propagation material. We developed an assay for use in indexing programs that is efficient and sensitive for detecting A. vitis in grape tissue. Initially, real-time polymerase chain reaction (PCR) primers specific for diverse tumorigenic strains of A. vitis were developed using the virD2 gene sequence. To overcome the effects of PCR inhibitors present in plant tissue, DNA extraction methods that included magnetic capture hybridization (MCH), immunomagnetic separation (IMS), and extraction with the Mo Bio Powerfood kit were compared. The assays incorporating MCH or IMS followed by real-time PCR were 10,000-fold more sensitive than direct real-time PCR when tested using boiled bacterial cell suspensions, with detection thresholds of 101 CFU/ml compared with 105 CFU/ml. DNA extraction with the Powerfood DNA extraction kit was 10-fold more sensitive than direct real-time PCR, with a detection threshold of 104 CFU/ml. All three assays were able to detect A. vitis in healthy-appearing grapevine cuttings taken from infected vines.

Critical Issues in Detecting Viable Listeria monocytogenes Cells by Real-Time Reverse Transcriptase PCR

2012 ◽  
Vol 75 (3) ◽  
pp. 512-517 ◽  
Author(s):  
LINLIN XIAO ◽  
LU ZHANG ◽  
HUA H. WANG
Keyword(s):  
Listeria Monocytogenes ◽  
16S Rrna ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Pcr Primers ◽  
Detection Methods ◽  
Rt Pcr ◽  
Heat Treated ◽  
Pcr Assays ◽  
The Impact

Rapid and specific detection of viable Listeria monocytogenes cells, particularly in processed foods, is a major challenge in the food industry. To assess the suitability of using RNA-based detection methods to detect viable cells, several sets of PCR primers and florescent probes were designed targeting the 16S rRNA, internalin A, and ribosomal protein L4 genes. One-step real-time reverse transcriptase (RT) PCR assays were conducted using RNAs extracted from control and heat-treated L. monocytogenes samples. The cycle threshold values were significantly higher in heat-treated cells than in controls. However, real-time RT-PCR amplification signals were still detected even in samples stored at room temperature for 24 h after lethal treatments, and the intensity of the signals was correlated with the cell population. The 16S rRNA molecules were the most stable of the three targets evaluated, and the impact on detection efficacy of the relative positions of the PCR primers within the target genes was limited under the experimental conditions. These results suggest that real-time RT-PCR assays have advantages over conventional PCR assays for assessing viable L. monocytogenes cells, but the results are affected by the stability of the RNA molecules targeted. These findings could have a major impact on interpretation of RNA-based detection data and gene expression studies.

scholarly journals Evaluation of Escherichia coli O157:H7 Growth Media for Use in Test-and-Hold Procedures for Ground Beef Processing†

2006 ◽  
Vol 69 (5) ◽  
pp. 1007-1011 ◽  
Author(s):  
MICHAEL N. GUERINI ◽  
TERRANCE M. ARTHUR ◽  
STEVEN D. SHACKELFORD ◽  
MOHAMMAD KOOHMARAIE
Keyword(s):  
Escherichia Coli ◽  
Time Course ◽  
Ground Beef ◽  
Threshold Level ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Growth Media ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Peptone Water

Since the mid-1990s, the beef industry has used a process called test and hold, wherein beef trim and ground beef are tested to keep products contaminated with Escherichia coli O157:H7 out of commerce. Current O157:H7 detection methods rely on a threshold level of bacterial growth for detection, which is dependent on the growth medium used. Twelve media were examined for growth and doubling time: buffered peptone water (BPW), SOC (which contains tryptone, yeast extract, KCl, MgCl2, and glucose), buffered peptone water plus SOC (BPW-SOC), Bacto-NZYM, RapidChek E. coli O157:H7 medium, BioControl EHEC8 culture medium, Neogen Reveal for E. coli O157:H7—Eight Hour medium (Neogen Reveal 8), BAX System medium for E. coli O157:H7 (BAX), BAX System medium for E. coli O157:H7 MP (BAX-MP), modified E. coli broth, nutrient medium, and tryptic soy broth (TSB). All media were tested at 37 or 42°C under static or shaking conditions. The eight media with the highest total CFU per milliliter and most rapid doubling times were BPW-SOC, NZYM, RapidChek, EHEC8, Neogen Reveal 8, BAX, BAX-MP, and TSB. The ability of these eight media to enrich E. coli O157:H7 in ground beef was further evaluated through time-course experiments using immunomagnetic separation. Of these media, TSB was the easiest to prepare, had a wide application base, and was the least expensive. In the test-and-hold process, the normal ratio of medium to product is 1:10. In this study, a 1:3 ratio worked as well as a 1:10 ratio. Processors using test-and-hold procedures could use 1 liter of TSB to enrich for E. coli O157:H7 in a 375-g sample instead of the usual 3.375 liters, thus saving reagents, time, and labor while maintaining accuracy.

Detection by real-time quaking-induced conversion (RT-QuIC), ELISA, and IHC of chronic wasting disease prion in lymph nodes from Pennsylvania white-tailed deer with specific PRNP genotypes

2021 ◽  
pp. 104063872110214
Author(s):  
Deepanker Tewari ◽  
David Steward ◽  
Melinda Fasnacht ◽  
Julia Livengood
Keyword(s):  
Real Time ◽  
Disease Susceptibility ◽  
Chronic Wasting Disease ◽  
Low Frequency ◽  
The United States ◽  
Detection Methods ◽  
Spongiform Encephalopathy ◽  
Wasting Disease ◽  
Diagnostic Laboratory ◽  
Highly Sensitive

Chronic wasting disease (CWD) is a prion-mediated, transmissible disease of cervids, including deer ( Odocoileus spp.), which is characterized by spongiform encephalopathy and death of the prion-infected animals. Official surveillance in the United States using immunohistochemistry (IHC) and ELISA entails the laborious collection of lymphoid and/or brainstem tissue after death. New, highly sensitive prion detection methods, such as real-time quaking-induced conversion (RT-QuIC), have shown promise in detecting abnormal prions from both antemortem and postmortem specimens. We compared RT-QuIC with ELISA and IHC for CWD detection utilizing deer retropharyngeal lymph node (RLN) tissues in a diagnostic laboratory setting. The RLNs were collected postmortem from hunter-harvested animals. RT-QuIC showed 100% sensitivity and specificity for 50 deer RLN (35 positive by both IHC and ELISA, 15 negative) included in our study. All deer were also genotyped for PRNP polymorphism. Most deer were homozygous at codons 95, 96, 116, and 226 (QQ/GG/AA/QQ genotype, with frequency 0.86), which are the codons implicated in disease susceptibility. Heterozygosity was noticed in Pennsylvania deer, albeit at a very low frequency, for codons 95GS (0.06) and 96QH (0.08), but deer with these genotypes were still found to be CWD prion-infected.

scholarly journals Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

Interlaboratory Validation of a Real-Time PCR 24-Hour Rapid Method for Detection of Salmonella in Foods

2009 ◽  
Vol 72 (5) ◽  
pp. 945-951 ◽  
Author(s):  
CHORNG-MING CHENG ◽  
KHANH T. VAN ◽  
WEN LIN ◽  
RICHARD M. RUBY
Keyword(s):  
Real Time ◽  
Screening Method ◽  
Rapid Screening ◽  
Selective Enrichment ◽  
Manual Method ◽  
Qpcr Method ◽  
Peptone Water ◽  
Interlaboratory Validation ◽  
Chili Powder ◽  
The One

The efficacy of a 24-h Salmonella real-time, or quantitative, PCR (qPCR) detection method was assessed through a collaborative effort involving eight Federal and state laboratories. Eleven foods including mashed potatoes, soft cheese, chili powder, chocolate, eggs, sprouts, apple juice, fish, shrimp, ground beef, and ground chicken were tested. For each food, seven blind samples were distributed to each participant for testing. These included six samples equivalently inoculated with 1 to 5 CFU/25 g of various serotypes of Salmonella (Gaminara, Weltevreden, Heidelberg, Senftenberg, Enteritidis, Newport, Typhimurium, and Kentucky for each food) and 10 to 50 CFU/25 g of the competitor Enterobacter cloacae. The seventh sample was inoculated with 10 to 50 CFU/25 g of the competitor, E. cloacae, only. These samples were tested for Salmonella by using four methods in parallel: (i) 24-h qPCR method detecting Salmonella from modified buffered peptone water enrichment medium; (ii) 48-h qPCR method detecting Salmonella from a secondary selective enrichment broth; (iii) modified Bacteriological Analytical Manual method; and (iv) VIDAS, an immunoassay system. The results of the statistical analysis showed there was no significant (P ≥ 0.05) difference between either of the qPCR methods and the modified Bacteriological Analytical Manual method for 10 of 11 foods. For the one exception, sprouts, detection by qPCR required 48 h. Both qPCR methods showed a detection limit of 0.08 to 0.2 CFU/g. These results provide a solid basis for using this 24-h qPCR rapid screening method to detect Salmonella in foods.

scholarly journals Recent Advances on Early Detection of Heat Strain in Dairy Cows Using Animal-Based Indicators: A Review

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 980
Author(s):  
Hang Shu ◽  
Wensheng Wang ◽  
Leifeng Guo ◽  
Jérôme Bindelle
Keyword(s):  
Heat Stress ◽  
Early Detection ◽  
Dairy Cows ◽  
Real Time ◽  
Dairy Farms ◽  
Time Measurement ◽  
Heat Strain ◽  
Detection Methods ◽  
Livestock Farming ◽  
Real Time Measurement

In pursuit of precision livestock farming, the real-time measurement for heat strain-related data has been more and more valued. Efforts have been made recently to use more sensitive physiological indicators with the hope to better inform decision-making in heat abatement in dairy farms. To get an insight into the early detection of heat strain in dairy cows, the present review focuses on the recent efforts developing early detection methods of heat strain in dairy cows based on body temperatures and respiratory dynamics. For every candidate animal-based indicator, state-of-the-art measurement methods and existing thresholds were summarized. Body surface temperature and respiration rate were concluded to be the best early indicators of heat strain due to their high feasibility of measurement and sensitivity to heat stress. Future studies should customize heat strain thresholds according to different internal and external factors that have an impact on the sensitivity to heat stress. Wearable devices are most promising to achieve real-time measurement in practical dairy farms. Combined with internet of things technologies, a comprehensive strategy based on both animal- and environment-based indicators is expected to increase the precision of early detection of heat strain in dairy cows.

scholarly journals Separating damage from environmental effects affecting civil structures for near real-time damage detection

2017 ◽  
Vol 17 (4) ◽  
pp. 850-868 ◽  
Author(s):  
William Soo Lon Wah ◽  
Yung-Tsang Chen ◽  
Gethin Wyn Roberts ◽  
Ahmed Elamin
Keyword(s):  
Damage Detection ◽  
Real Time ◽  
Environmental Effects ◽  
Environmental Conditions ◽  
Principal Component ◽  
Detection Methods ◽  
Real Time Monitoring ◽  
Civil Structures ◽  
Data Set ◽  
Wide Range

Analyzing changes in vibration properties (e.g. natural frequencies) of structures as a result of damage has been heavily used by researchers for damage detection of civil structures. These changes, however, are not only caused by damage of the structural components, but they are also affected by the varying environmental conditions the structures are faced with, such as the temperature change, which limits the use of most damage detection methods presented in the literature that did not account for these effects. In this article, a damage detection method capable of distinguishing between the effects of damage and of the changing environmental conditions affecting damage sensitivity features is proposed. This method eliminates the need to form the baseline of the undamaged structure using damage sensitivity features obtained from a wide range of environmental conditions, as conventionally has been done, and utilizes features from two extreme and opposite environmental conditions as baselines. To allow near real-time monitoring, subsequent measurements are added one at a time to the baseline to create new data sets. Principal component analysis is then introduced for processing each data set so that patterns can be extracted and damage can be distinguished from environmental effects. The proposed method is tested using a two-dimensional truss structure and validated using measurements from the Z24 Bridge which was monitored for nearly a year, with damage scenarios applied to it near the end of the monitoring period. The results demonstrate the robustness of the proposed method for damage detection under changing environmental conditions. The method also works despite the nonlinear effects produced by environmental conditions on damage sensitivity features. Moreover, since each measurement is allowed to be analyzed one at a time, near real-time monitoring is possible. Damage progression can also be given from the method which makes it advantageous for damage evolution monitoring.

Design on Experience Virtual Tour System

2014 ◽  
Vol 536-537 ◽  
pp. 603-606
Author(s):  
Yu Mei Liu ◽  
Yu Dan Dong ◽  
Jing Wu
Keyword(s):  
Real Time ◽  
Collision Detection ◽  
Detection Methods ◽  
Model Structure ◽  
Object Model ◽  
Tourist Attractions ◽  
Virtual Tour ◽  
Modeling Techniques

According to the characteristics and needs of virtual scenic roaming system, select the appropriate modeling techniques. By using the modeling platform scenic entity object model structure, and then build virtual tourist attractions, we propose hierarchical collision detection methods. This method actually meets the accuracy requirements under the premise, greatly reducing the number and complexity of collision detection; effectively improve the system in real time.

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