scholarly journals Evaluation of Air Sampling and Detection Methods to Quantify Airborne Ascospores of Sclerotinia sclerotiorum

Plant Disease ◽  
2014 ◽  
Vol 98 (1) ◽  
pp. 32-42 ◽  
Author(s):  
M. L. Parker ◽  
M. R. McDonald ◽  
G. J. Boland
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Quantitative Polymerase Chain Reaction ◽  
Air Sampling ◽  
Forecast Model ◽  
Qpcr Assay ◽  
Detection Methods ◽  
Air Samples ◽  
Growing Seasons ◽  
Burkard Sampler ◽  
Cyclone Sampler

Detection and quantification of airborne ascospores as a component of the Sclerotinia rot of carrot (SRC) forecast model is currently accomplished using the blue plate test (BPT), which uses Sclerotinia semiselective medium (SSM). A quantitative polymerase chain reaction (qPCR) assay was developed to reduce the time to specifically quantify ascospores of Sclerotinia sclerotiorum from air samples collected using a Burkard Multi-Vial Cyclone Sampler. The qPCR assay was highly sensitive and detected DNA from 0.5 to 5 × 104 ascospores within a linear range (R2 = 0.99). The qPCR assay was used to quantify ascospores of S. sclerotiorum in air samples collected over three growing seasons. Initial SRC disease was observed 8 and 34 days following detection of 9.5 and 2 ascospores m–3 of air, respectively. Results from air samples collected using an Andersen N6 Sampler and the qPCR assay were compared with the BPT. Ascospore counts from a Burkard Sampler coupled with the qPCR assay and the BPT followed similar trends. In general, fewer ascospores were detected and bioaerosol sampling efficiency was low using an Anderson Sampler. Three days were required to confirm the number of ascospores using SSM in the BPT and with an Andersen Sampler, whereas results from a Burkard Sampler coupled with the qPCR assay can provide results within 5 h of air sampling. The choice of method will depend on the available resources.

scholarly journals A Quantitative PCR System for Measuring Sclerotinia sclerotiorum in Canola (Brassica napus)

Plant Disease ◽  
2016 ◽  
Vol 100 (5) ◽  
pp. 984-990 ◽  
Author(s):  
B. R. Ziesman ◽  
T. K. Turkington ◽  
U. Basu ◽  
S. E. Strelkov
Keyword(s):  
Risk Assessment ◽  
Brassica Napus ◽  
Sclerotinia Sclerotiorum ◽  
Assessment Tool ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Stem Rot ◽  
Specific Gene ◽  
Sclerotinia Stem Rot ◽  
Hydrolysis Probe

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is an economically important disease of canola (Brassica napus) commonly managed by routine application of fungicides. Petal infestation has been demonstrated to be an important stage of the disease cycle in canola and has been the focus of previously developed Sclerotinia stem rot risk assessment methods. Quantitative polymerase chain reaction (qPCR) analysis can provide a more rapid and accurate assessment of petal infestation levels. Primers and a hydrolysis probe were designed to amplify a 70-bp region of an S. sclerotiorum-specific gene, SS1G_00263. A hydrolysis probe-based qPCR assay was developed that had a detection limit of 8.0 × 10−4 ng of S. sclerotiorum DNA and only amplified S. sclerotiorum DNA. Evaluation of petals collected at five sampling points in each of 10 commercial canola fields on each of two sampling dates (corresponding to 20 to 30% bloom and 40 to 50% bloom) revealed S. sclerotiorum DNA infestation levels of 0 to 3.3 × 10−1 ng/petal. This qPCR assay can be used to reliably quantify petal infestation and, with further research, has the potential to serve as the basis for a Sclerotinia stem rot risk assessment tool or as a means to study Sclerotinia stem rot epidemiology.

Comparison between the Real-Time PCR and Crystal Diagnostic Xpress Immunoassay Methods for Detecting Salmonella and Shiga Toxin–Producing Escherichia coli in the Air of Beef Slaughter Establishments

2020 ◽  
Vol 84 (1) ◽  
pp. 31-38
Author(s):  
ZAHRA MOHAMMAD ◽  
SAMUEL BECK ◽  
MARIA KING ◽  
DAVEY GRIFFIN ◽  
ALEJANDRO CASTILLO
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Qpcr Assay ◽  
Detection Methods ◽  
Processing Plant ◽  
Serological Tests ◽  
Detection Rates ◽  
Air Samples

ABSTRACT The aim of this study was to compare the effectiveness of a quantitative real-time PCR (qPCR) molecular method and the Crystal Diagnostic Xpress (CDx) immunoassay for detecting Salmonella and Shiga toxin–producing Escherichia coli (STEC) in air samples collected from abattoirs in Texas. The 70 air samples were collected from two small and two large meat processing plants in the spring and summer with a wetted wall cyclone air sampler. The samples were divided equally into two parts: one part was used for the qPCR assay, and the other part was enriched for 18 and 36 h and evaluated with the CDx immunoassay. All samples for which positive results were obtained were confirmed by plating and by biochemical and serological tests as recommended by AOAC International to verify results of rapid methods. With the qPCR and CDx assays and 36 h of enrichment, 37.5 and 57.1% of the samples, respectively, were positive for Salmonella (P < 0.05) and 65.0 and 60.7%, respectively, were positive for STEC (P > 0.05). Air samples required longer enrichment for the CDx immunoassay than recommended by the manufacturer for food samples. Recovery of Salmonella and STEC increased 16 and 47%, respectively, when enrichment was extended from 18 to 36 h. The prevalence of Salmonella and STEC obtained with both methods was affected by the size of the processing plant and the processing stage. Detection rates for samples from larger plants were higher for both pathogens. Significantly higher prevalence was obtained for samples from the stunning and dehiding areas than for those from the fabrication rooms and chillers. Salmonella detection was higher with the CDx assay than with the qPCR assay, but no differences were found for the detection of STEC by the qPCR and CDx assays. These results highlight the importance of method adjustments when testing matrices other than foods. More research is needed to understand the dynamics of pathogen dispersal in aerosols and how this affects the effectiveness of current rapid detection methods. HIGHLIGHTS

Effect of Building Construction on Aspergillus Concentrations in a Hospital

2008 ◽  
Vol 29 (5) ◽  
pp. 462-464 ◽  
Author(s):  
Marian D. Goebes ◽  
Ellen Jo Baron ◽  
Kathleen L. Mathews ◽  
Lynn M. Hildemann
Keyword(s):  
Polymerase Chain Reaction ◽  
Particulate Matter ◽  
Negative Pressure ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Building Construction ◽  
Chain Reaction ◽  
Air Samples ◽  
Polymerase Chain ◽  
Construction Zone

Air samples taken in a hospital undergoing construction and analyzed with a quantitative polymerase chain reaction (qPCR) assay for the Aspergillus genus did not show elevated concentrations of Aspergillus or particulate matter with a diameter of 5 μm or less in patient areas. Air samples from the construction zone indicated the containment system, which used polyethylene film barrier and negative pressure, was effective.

scholarly journals Eliminating false positives in a qPCR assay for the detection of the uidA gene in Escherichia coli

2013 ◽  
Vol 11 (3) ◽  
pp. 382-386 ◽  
Author(s):  
Richard Kibbee ◽  
Natalie Linklater ◽  
Banu Örmeci
Keyword(s):  
Escherichia Coli ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Uida Gene ◽  
Chain Reaction ◽  
Taq Polymerase ◽  
E Coli ◽  
Gene Copies ◽  
Polymerase Chain ◽  
Low Levels

Due to contaminant Escherichia coli DNA present in recombinant Taq polymerase reagents, it is not possible to reliably detect low levels of E. coli in samples using the quantitative polymerase chain reaction (qPCR) assay. Native Taq polymerase was successfully used in this study to detect five uidA gene copies (5 fg of genomic DNA) of the uidA gene.

scholarly journals Development and Application of a Quantitative PCR Detection Method to Quantify Venturia oleaginea in Asymptomatic Olive (Olea europaea) Leaves

2020 ◽  
Vol 110 (3) ◽  
pp. 547-555
Author(s):  
Silvia Scibetta ◽  
Giovanni E. Agosteo ◽  
Ahmed Abdelfattah ◽  
Maria G. Li Destri Nicosia ◽  
Santa O. Cacciola ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
High Throughput Sequencing ◽  
Current Knowledge ◽  
Leaf Growth ◽  
Its Region ◽  
Qpcr Assay ◽  
Detection Methods ◽  
Effective Control ◽  
Its2 Region

Olive leaf spot (OLS), caused by Venturia oleaginea, is one of the most common and serious diseases of olive trees in the Mediterranean region. Understanding the pathogen life cycle is important for the development of effective control strategies. Current knowledge is incomplete owing to a lack of effective detection methods. It is extremely difficult to culture V. oleaginea in vitro, so primers were designed to amplify and sequence the internal transcribed spacer ITS1-5.8S-ITS2 region of the fungus directly from infected olive leaves. Sanger sequencing indicated a unique ITS region present in the European strains screened, confirming the appropriateness of the target region for developing a quantitative PCR (qPCR) assay. Furthermore, high-throughput sequencing of the same region excluded the presence of other Venturia species in the olive phyllosphere. The qPCR assay proved very specific and sensitive, enabling the detection of approximately 26 copies of target DNA. The analysis of symptomless leaves during early stages of the epidemic from the end of winter through spring revealed a similar quantity of pathogen DNA regardless of the leaf growth stage. In contrast, the pathogen titer changed significantly during the season. Data indicated that leaf infections start earlier than expected over the season and very young leaves are as susceptible as adult leaves. These findings have important practical implications and suggest the need for improved scheduling of fungicide treatments. The qPCR assay represents a valuable tool providing quantitative results and enables detection of V. oleaginea in all olive organs, including those in which OLS cannot be studied using previously available methods.

scholarly journals qPCR Assays for the Detection of Cylindrocladium buxicola in Plant, Water, and Air Samples

Plant Disease ◽  
2013 ◽  
Vol 97 (8) ◽  
pp. 1082-1090 ◽  
Author(s):  
B. Gehesquière ◽  
S. D'Haeyer ◽  
K. T. K. Pham ◽  
A. J. Van Kuik ◽  
M. Maes ◽  
...  
Keyword(s):  
Water Samples ◽  
Western Europe ◽  
Extraction Methods ◽  
Detection Methods ◽  
Taqman Probe ◽  
Future Research ◽  
Plant Water ◽  
Long Distance ◽  
Air Samples ◽  
Latently Infected

Cylindrocladium buxicola (syn. C. pseudonaviculatum; teleomorph Calonectria pseudonaviculata) is an important fungal pathogen of Buxus spp. Although widespread in Western Europe, this pathogen has only recently been introduced into North America, where it represents a significant threat to the U.S. and Canadian boxwood industries. Trade of latently infected nursery stock is an important mode of long-distance dissemination and introduction of this pathogen but no methods for detection of latently infected material are available. Also, the pathways for short-distance dispersal of C. buxicola have not been adequately studied. Improved detection methods of this pathogen in air and water samples would benefit future research in this area. We have developed real-time polymerase chain reaction assays for the detection of C. buxicola based on the ribosomal DNA internal transcribed spacer 1 (ITS) and the β-tubulin 2 gene (TUB). Using a TaqMan probe conjugated with a 3′ minor groove binding group (TaqMan MGB probe), the ITS-based assay could reliably detect as little as 10 fg of genomic DNA or 20 copies of cloned target DNA and was approximately 70 times more sensitive than the SYBR Green TUB-based assay. The ITS-based assay provided good but not complete specificity, and is well suited for epidemiological studies. The TUB-based assay, however, proved to be fully specific and can be used for diagnostics. We developed and optimized sample processing and DNA extraction methods for detection of latently present C. buxicola in boxwood plants and quantification of conidia in water and air samples. C. buxicola could be detected in 20 g of plant material, of which only 1 ppm of the tissue was infected, in 10-ml water samples containing as low as 1 conidium/ml, and on Melinex tape pieces representing 12 h of air sampling containing 10 or more conidia. The applicability of the techniques to plant, water, and air samples of practical size was demonstrated.

scholarly journals Development of a Genus-Specific Brucella Real-Time PCR Assay Targeting the 16S-23S rDNA Internal Transcribed Spacer from Different Specimen Types

2020 ◽  
Vol 7 (4) ◽  
pp. 175
Author(s):  
Rejoice Nyarku ◽  
Ayesha Hassim ◽  
Annelize Jonker ◽  
Melvyn Quan
Keyword(s):  
Sensitivity And Specificity ◽  
Internal Transcribed Spacer ◽  
Bacterial Culture ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Surface Protein ◽  
Qpcr Assay ◽  
Rapid Screening ◽  
Analytical Sensitivity ◽  
Tissue Samples

The aim of this study was to develop a 16S-23S ribosomal deoxyribonucleic acid internal transcribed spacer (ITS) quantitative polymerase chain reaction (qPCR) assay for the early diagnosis and rapid screening of brucellosis. Blood, milk, and tissue samples were spiked with B. abortus biovar 1 (B01988-18 strain) to determine the analytical sensitivity and specificity of the assay. The 95% limit of detection of the ITS qPCR assay was highest in tissue, followed by blood, then milk, i.e., 0.48, 4.43, and 15.18 bacteria/PCR reaction, respectively. The diagnostic performance of the assay was compared to the Brucella cell surface protein (BCSP) 31 qPCR assay and bacterial culture. Out of 56 aborted foetal tissue samples from bovine, ovine, and caprine, 33% (19/56) were positive for Brucella spp. The sensitivity and specificity of the ITS qPCR assay was 87% and 95% respectively, compared to 92% and 89% for the BCSP31 qPCR assay and 47% and 55% for bacterial culture, respectively. The assay was efficient, sensitive, and specific, making it a valuable tool in the early detection of the Brucella pathogen.

Sign in / Sign up

Export Citation Format

Share Document