scholarly journals Real-time quantitative PCR for enteric adenovirus serotype 40 in environmental waters

2005 ◽  
Vol 51 (5) ◽  
pp. 393-398 ◽  
Author(s):  
Sunny Jiang ◽  
Hojabr Dezfulian ◽  
Weiping Chu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Amplification Efficiency ◽  
Sewage Effluent ◽  
Hexon Gene ◽  
Pcr Assay ◽  
Environmental Waters ◽  
Cycle Number ◽  
The Real

Adenoviruses 40 and 41 have been recognized as important etiological agents of gastroenteritis in children. A real-time PCR method (TaqMan® assay) was developed for rapid quantification of adenovirus 40 (Ad40) by amplifying an 88 bp sequence from the hexon gene. To establish a quantification standard curve, a 1090 bp hexon region of Ad40 was amplified and cloned into the pGEM®-T Vector. A direct correlation was observed between the fluorescence threshold cycle number (Ct) and the starting quantity of Ad40 hexon gene. The quantification was linear over 6-log units and the amplification efficiency averaged greater than 95%. Seeding studies using various environmental matrices (including sterile water, creek water, brackish estuarine water, ocean water, and secondary sewage effluent) suggest that this method is applicable to environmental samples. However, real-time PCR was sensitive to inhibitors present in the environmental samples. Lower efficiency of PCR amplification was found in secondary sewage effluent and creek waters. Application of the method to fecal contaminated waters successfully quantified the presence of Ad40. The sensitivity of the real-time PCR is comparable to the traditional nested PCR assay for environmental samples. In addition, the real-time PCR assay offers the advantage of speed and insensitivity to contamination during PCR set up. The real-time PCR assay developed in this study is suitable for quantitative determination of Ad40 in environmental samples and represents a considerable advancement in pathogen quantification in aquatic environments.Key words: adenovirus, real-time PCR, environmental waters, serotype 40.

scholarly journals Real-Time PCR Assay for the Detection and Quantification of Roe Deer to Detect Food Adulteration—Interlaboratory Validation Involving Laboratories in Austria, Germany, and Switzerland

Foods ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2645
Author(s):  
Barbara Druml ◽  
Steffen Uhlig ◽  
Kirsten Simon ◽  
Kirstin Frost ◽  
Karina Hettwer ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Roe Deer ◽  
Amplification Efficiency ◽  
Pcr Assay ◽  
Food Adulteration ◽  
The Real ◽  
Game Meat ◽  
Meat Samples ◽  
Detection And Quantification

Game meat products are particularly prone to be adulterated by replacing game meat with cheaper meat species. Recently, we have presented a real-time polymerase chain reaction (PCR) assay for the identification and quantification of roe deer in food. Quantification of the roe deer content in % (w/w) was achieved relatively by subjecting the DNA isolates to a reference real-time PCR assay in addition to the real-time PCR assay for roe deer. Aiming at harmonizing analytical methods for food authentication across EU Member States, the real-time PCR assay for roe deer has been tested in an interlaboratory ring trial including 14 laboratories from Austria, Germany, and Switzerland. Participating laboratories obtained aliquots of DNA isolates from a meat mixture containing 24.8% (w/w) roe deer in pork, roe deer meat, and 12 meat samples whose roe deer content was not disclosed. Performance characteristics included amplification efficiency, level of detection (LOD95%), repeatability, reproducibility, and accuracy of quantitative results. With a relative reproducibility standard deviation ranging from 13.35 to 25.08% (after outlier removal) and recoveries ranging from 84.4 to 114.3%, the real-time PCR assay was found to be applicable for the detection and quantification of roe deer in raw meat samples to detect food adulteration.

Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

2012 ◽  
Vol 75 (4) ◽  
pp. 743-747 ◽  
Author(s):  
BWALYA LUNGU ◽  
W. DOUGLAS WALTMAN ◽  
ROY D. BERGHAUS ◽  
CHARLES L. HOFACRE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

2007 ◽  
Vol 70 (5) ◽  
pp. 1080-1087 ◽  
Author(s):  
V. M. BOHAYCHUK ◽  
G. E. GENSLER ◽  
M. E. McFALL ◽  
R. K. KING ◽  
D. G. RENTER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Conventional Culture ◽  
Food Animal ◽  
Highly Sensitive ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

scholarly journals Susceptibility of Maize Inbreds and Incidence of Symptomless Infection by the Head Smut Pathogen, Sphacelotheca reiliana

2016 ◽  
Vol 17 (1) ◽  
pp. 1-5 ◽  
Author(s):  
S. J. Anderson ◽  
H. E. Simmons ◽  
R. D. French-Monar ◽  
G. P. Munkvold
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Field Trials ◽  
Growth Stages ◽  
High Susceptibility ◽  
Head Smut ◽  
Pcr Assay ◽  
Disease Symptoms ◽  
The Real ◽  
Seedling Infection

A real-time PCR assay was used to compare seedling infection by Sphacelotheca reiliana, the causal agent of head smut, among five inbred genotypes representing low, moderate, and high susceptibility to the disease. Seeds were coated with teliospores and planted in autoclaved field soil in a growth chamber. Incidence of seedling infection at growth stage V3 differed between an inbred genotype of low susceptibility and those of moderate and high susceptibility, but did not differ between the high and moderately susceptible groups (P < 0.05). The real-time PCR assay was also used to compare infection status at early and late vegetative stages with observable symptoms in the field. We detected infection via real-time PCR in maize at both growth stages during field trials conducted in Texas and California but observed no disease symptoms (smutted ears or tassels). Notably, the fungus was present in up to 31% of the ear shoots in plots without disease symptoms. The real-time assay can be a useful tool for screening seedling-stage host resistance, and for better understanding the progress of infection in different maize genotypes. The field data suggest that asymptomatic infection is much more common than previously thought, and may have important implications for the epidemiology of this fungus under diverse plant resistance and growing conditions. Accepted for publication 11 December 2015. Published 5 January 2016.

Real-Time PCR Quantification of Vibrio parahaemolyticus in Oysters Using an Alternative Matrix

2004 ◽  
Vol 67 (11) ◽  
pp. 2424-2429 ◽  
Author(s):  
G. E. KAUFMAN ◽  
G. M. BLACKSTONE ◽  
M. C. L. VICKERY ◽  
A. K. BEJ ◽  
J. BOWERS ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Pcr Amplification ◽  
Oyster Tissue ◽  
Pcr Assay ◽  
Colony Hybridization ◽  
The Real ◽  
Mantle Fluid ◽  
June July

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26°C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P &lt; 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P &lt; 0.05; and oyster: r = 0.99, P &lt; 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P &lt; 0.05) but reduced correlation (r =−0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus–specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.

Detection and Enumeration of Salmonella Enteritidis in Homemade Ice Cream Associated with an Outbreak: Comparison of Conventional and Real-Time PCR Methods

2006 ◽  
Vol 69 (3) ◽  
pp. 639-643 ◽  
Author(s):  
K. H. SEO ◽  
I. E. VALENTIN-BON ◽  
R. E. BRACKETT
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Direct Detection ◽  
Ice Cream ◽  
Plate Count ◽  
Pcr Assay ◽  
The Real ◽  
Real Time Quantitative Pcr

Salmonellosis caused by Salmonella Enteritidis (SE) is a significant cause of foodborne illnesses in the United States. Consumption of undercooked eggs and egg-containing products has been the primary risk factor for the disease. The importance of the bacterial enumeration technique has been enormously stressed because of the quantitative risk analysis of SE in shell eggs. Traditional enumeration methods mainly depend on slow and tedious most-probable-number (MPN) methods. Therefore, specific, sensitive, and rapid methods for SE quantitation are needed to collect sufficient data for risk assessment and food safety policy development. We previously developed a real-time quantitative PCR assay for the direct detection and enumeration of SE and, in this study, applied it to naturally contaminated ice cream samples with and without enrichment. The detection limit of the real-time PCR assay was determined with artificially inoculated ice cream. When applied to the direct detection and quantification of SE in ice cream, the real-time PCR assay was as sensitive as the conventional plate count method in frequency of detection. However, populations of SE derived from real-time quantitative PCR were approximately 1 log higher than provided by MPN and CFU values obtained by conventional culture methods. The detection and enumeration of SE in naturally contaminated ice cream can be completed in 3 h by this real-time PCR method, whereas the cultural enrichment method requires 5 to 7 days. A commercial immunoassay for the specific detection of SE was also included in the study. The real-time PCR assay proved to be a valuable tool that may be useful to the food industry in monitoring its processes to improve product quality and safety.

scholarly journals Real-Time PCR Improves Detection ofTrichomonas vaginalisInfection Compared With Culture Using Self-Collected Vaginal swabs

2005 ◽  
Vol 13 (3) ◽  
pp. 145-150 ◽  
Author(s):  
A. M. Caliendo ◽  
J. A. Jordan ◽  
A. M. Green ◽  
J. Ingersoll ◽  
R. J. Diclemente ◽  
...  
Keyword(s):  
African American Women ◽  
Real Time ◽  
Real Time Pcr ◽  
Trichomonas Vaginalis ◽  
Repeated Sequence ◽  
Adolescent And Young Adult ◽  
Broth Culture ◽  
Pcr Assay ◽  
The Real ◽  
Vaginal Swabs

Objective.To compare a real-time polymerase chain reaction (PCR) assay with broth culture for the detection ofTrichomonas vaginalisusing self-collected vaginal swabs.Methods.Self-collected vaginal swabs were obtained from adolescent and young adult African-American women participating in HIV-1 prevention programs.T. vaginalisculture was performed using the InPouch TV System. Samples for the real-time PCR assay were collected using the BDProbeTec ET Culturette Direct Dry Swab system and tested in a laboratory-developed assay which targeted a repeated sequence of the genome. Discrepant samples that were culture negative and positive in the real-time PCR assay were tested in a confirmatory PCR which targeted a different region of theT. vaginalisgenome, the18S ribosomal DNA gene.Results.Of the 524 specimens tested by both culture and real-time PCR, 36 were culture positive and 54 were positive in the real-time PCR assay; 16 of the 18 discrepant specimens were also positive in the confirmatory PCR assay. Using a modified gold standard of positive by culture or positive in both PCR assays, the sensitivity of the real-time PCR assay was 100% and the specificity was 99.6%, whereas culture had a sensitivity of 69.2% and a specificity of 100%.Conclusions.The real-time PCR assay was sensitive and specific for the detection ofT. vaginalisDNA from self-collected vaginal swab specimens. The ability to use the BDProbeTec dry swab system for the real-time PCR testing allowed for the detection ofChlamydia trachomatis, Neisseria gonorrhoeae,andT. vaginalisfrom a single specimen.

Real-Time Polymerase Chain Reaction Detection of Bovine DNA in Meat and Bone Meal Samples

2002 ◽  
Vol 65 (7) ◽  
pp. 1158-1165 ◽  
Author(s):  
S. LAHIFF ◽  
M. GLENNON ◽  
J. LYNG ◽  
T. SMITH ◽  
N. SHILTON ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

We describe a real-time polymerase chain reaction (PCR) assay for the detection of bovine DNA extracted from meat and bone meal (MBM) samples. PCR primers were used to amplify a 271-bp region of the mitochondrial ATPase 8–ATPase 6 gene, and a fluorogenic probe (BOV1) labeled with a 5′ FAM reporter and a 3′ TAMRA quencher was designed to specifically detect bovine PCR product. The specificity of the BOV1 probe for the detection of the bovine PCR product was confirmed by Southern blot hybridization analysis of the probe with PCR products generated from ovine, porcine, and bovine genomic DNA extracted from blood and with PCR products generated from genomic DNA extracted from single-species laboratory scale rendered MBM samples. The specificity of the BOV1 probe was also evaluated in real-time PCR reactions including these genomic targets. Both methods demonstrated that the BOV1 probe was specific for the detection of bovine PCR product. The BOV1 probe had a detection limit of 0.0001% bovine material by Southern blot DNA probe hybridization analysis and a detection limit of 0.001% bovine material in the real-time PCR assay. Application of the real-time PCR assay to six industrial samples that had previously tested positive for the presence of bovine material with a conventional PCR assay yielded positive results with the real-time PCR assay for four samples.

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