Specificity and sensitivity of three PCR-based methods for detection of Erwinia amylovora in pure culture and plant material

Three PCR methods, referred in this study as ?conventional?, ?nested? and ?chromosomal? PCR and suggested for routine detection of Erwinia amylovora in pure culture and plant material, were evaluated according to their specificity and sensitivity. Specificity of PCR methods was analyzed by using 42 strains of E. amylovora, originating from different locations and plant species, with diverse PFGE profiles, representing distant populations of the pathogen. Sensitivity of PCR protocols in pure culture was studied by using nine different concentrations of E. amylovora in sterile ultrapure water as a template in PCR reactions. In order to study inhibitory effect of plant DNA and other inhibitors on sensitivity of the three PCR methods bacterial dilutions were mixed with plant macerate of pear, apple and quince prior to the PCR reaction. In specificity assays, tested PCR protocols were able to detect all E. amylovora strains regardless of the host of the strain, its origin or PFGE group, indicating primer specificity. On the other hand, sensitivity among tested methods varied, depending on bacterial concentration and selected plant material used in the PCR. When working with pure cultures nested PCR showed the greatest sensitivity by detecting 1.9 bacterial cells per PCR reaction, followed by detection limit of 9.5 cells per PCR reaction with conventional PCR and 1.9?105 cells/PCR reaction with chromosomal PCR. In spiked samples plant inhibitors either did not affect or they decreased the sensitivity of the PCR reaction, depending on the protocol and/or type of plant macerate. In our experiments, inhibitors from pear and quince macerates did not affect sensitivity of nested PCR, while apple macerate reduced its sensitivity by a factor of 10. Conventional PCR protocol was able to detect 95 cells/PCR reaction in pear and apple macerate, but only 9.5?103 cells/PCR in quince macerate. Greatest decrease in sensitivity of the PCR method was observed in spiked samples with chromosomal PCR since bacterial DNA was not detected in each of the spiked samples. Our research shows that all three PCR protocols are specific for detection of E. amylovora, but nested PCR proved to be most sensitive when working with pure cultures and plant material.

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Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovorain Asymptomatic Plant Material

Applied and Environmental Microbiology ◽

10.1128/aem.66.5.2071-2078.2000 ◽

2000 ◽

Vol 66 (5) ◽

pp. 2071-2078 ◽

Cited By ~ 84

Author(s):

Pablo Llop ◽

Anna Bonaterra ◽

Javier Peñalver ◽

María M. López

Keyword(s):

Nested Pcr ◽

Plant Material ◽

High Sensitivity ◽

Epidemiological Studies ◽

Sample Volume ◽

Small Sample ◽

Asymptomatic Plant ◽

Specificity And Sensitivity ◽

Closed Tube ◽

Pure Cultures

ABSTRACT A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 μl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.

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Conventional PCR for detection of Corynespora cassiicola in soybean seeds

Journal of Seed Science ◽

10.1590/2317-1545v38n2152049 ◽

2016 ◽

Vol 38 (2) ◽

pp. 85-91 ◽

Cited By ~ 5

Author(s):

Marcella Viana de Sousa ◽

Carolina da Silva Siqueira ◽

José da Cruz Machado

Keyword(s):

Pure Culture ◽

Early Stage ◽

Soybean Seeds ◽

Inoculum Potential ◽

Corynespora Cassiicola ◽

Severe Damage ◽

Conventional Pcr ◽

Seed Testing ◽

Target Spot ◽

Specificity And Sensitivity

Abstract The fungus Corynespora cassiicola, causal agent of target spot in soybeans, can be transmitted by soybean seeds and as of that point cause severe damage. This disease may be diagnosed at an early stage by seed testing, but knowledge in this area is insufficient. Because of that and increased attack by the disease in soybean areas in Brazil, further studies are required. The aim of this study was to evaluate the use of conventional PCR in detecting C. cassiicola in soybean seeds. The GA4-F/GA4-R primers described in the literature were tested for their specificity and sensitivity for detection of C. cassiicola in pure culture and in soybean seeds. Uninoculated and inoculated seed samples were used with different incidence levels - 100%, 10%, 1%, 0.5%, 0.25%, and 0% of preestablished inoculum potentials, P0, P1, P2, and P3. Detection of C. cassiicola in P1 inoculum potential was observed in samples with incidence levels of 10% to 100%. In the P3 potential, detection of the pathogen was successful in samples at the low level of 0.25%.

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Development of a Co-Dominant Cleaved Amplified Polymorphic Sequences Assay for the Rapid Detection and Differentiation of Two Pathogenic Clarireedia spp. Associated with Dollar Spot in Turfgrass

Agronomy ◽

10.3390/agronomy11081489 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1489

Author(s):

Tammy Stackhouse ◽

Sumyya Waliullah ◽

Alfredo D. Martinez-Espinoza ◽

Bochra Bahri ◽

Emran Ali

Keyword(s):

Pcr Primers ◽

The United States ◽

Fungal Species ◽

Dollar Spot ◽

Direct Pcr ◽

Specific Pcr ◽

Specificity And Sensitivity ◽

Pure Cultures ◽

Speed Up ◽

Cleaved Amplified Polymorphic Sequences

Dollar spot is one of the most destructive diseases in turfgrass. The causal agents belong to the genus Clarireedia, which are known for causing necrotic, sunken spots in turfgrass that coalesce into large damaged areas. In low tolerance settings like turfgrass, it is of vital importance to rapidly detect and identify the pathogens. There are a few methods available to identify the genus Clarireedia, but none of those are rapid enough and characterize down to the species level. This study produced a co-dominant cleaved amplified polymorphic sequences (CAPS) test that differentiates between C. jacksonii and C. monteithiana, the two species that cause dollar spot disease within the United States. The calmodulin gene (CaM) was targeted to generate Clarireedia spp. specific PCR primers. The CAPS assay was optimized and tested for specificity and sensitivity using DNA extracted from pure cultures of two Clarireedia spp. and other closely related fungal species. The results showed that the newly developed primer set could amplify both species and was highly sensitive as it detected DNA concentrations as low as 0.005 ng/µL. The assay was further validated using direct PCR to speed up the diagnosis process. This drastically reduces the time needed to identify the dollar spot pathogens. The resulting assay could be used throughout turfgrass settings for a rapid and precise identification method in the US.

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A Real-Time Thermal Sensor System for Quantifying the Inhibitory Effect of Antimicrobial Peptides on Bacterial Adhesion and Biofilm Formation

Sensors ◽

10.3390/s21082771 ◽

2021 ◽

Vol 21 (8) ◽

pp. 2771

Author(s):

Tobias Wieland ◽

Julia Assmann ◽

Astrid Bethe ◽

Christian Fidelak ◽

Helena Gmoser ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Real Time ◽

Bacterial Adhesion ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Bovine Mastitis ◽

Inhibitory Effect ◽

Thermal Sensor ◽

Bacterial Cells ◽

Static Conditions

The increasing rate of antimicrobial resistance (AMR) in pathogenic bacteria is a global threat to human and veterinary medicine. Beyond antibiotics, antimicrobial peptides (AMPs) might be an alternative to inhibit the growth of bacteria, including AMR pathogens, on different surfaces. Biofilm formation, which starts out as bacterial adhesion, poses additional challenges for antibiotics targeting bacterial cells. The objective of this study was to establish a real-time method for the monitoring of the inhibition of (a) bacterial adhesion to a defined substrate and (b) biofilm formation by AMPs using an innovative thermal sensor. We provide evidence that the thermal sensor enables continuous monitoring of the effect of two potent AMPs, protamine and OH-CATH-30, on surface colonization of bovine mastitis-associated Escherichia (E.) coli and Staphylococcus (S.) aureus. The bacteria were grown under static conditions on the surface of the sensor membrane, on which temperature oscillations generated by a heater structure were detected by an amorphous germanium thermistor. Bacterial adhesion, which was confirmed by white light interferometry, caused a detectable amplitude change and phase shift. To our knowledge, the thermal measurement system has never been used to assess the effect of AMPs on bacterial adhesion in real time before. The system could be used to screen and evaluate bacterial adhesion inhibition of both known and novel AMPs.

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Occurrence of Bacterial Stem Rot, Caused by Erwinia chrysanthemi, on Field-Grown Tomato in Florida

Plant Disease ◽

10.1094/pdis.1998.82.7.831c ◽

1998 ◽

Vol 82 (7) ◽

pp. 831-831 ◽

Cited By ~ 1

Author(s):

D. O. Chellemi ◽

H. A. Dankers ◽

K. Hill ◽

R. E. Cullen ◽

G. W. Simone ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Methyl Esters ◽

Sole Source ◽

Erwinia Chrysanthemi ◽

Identification System ◽

Bacterial Cells ◽

Sterile Water ◽

Microbial Identification ◽

Pure Cultures ◽

Tomato Isolate

In September 1997, wilted 4-week-old tomato (Lycopersicon esculentum Mill.) plants were observed in a commercial production field in St. Lucie County, FL. Closer inspection of affected plants revealed hollow stems and petioles with dark, water-soaked lesions. Diseased tissue was macerated and streaked onto nutrient agar (NA) and crystal violet pectate (CVP) agar. After incubation for 2 days at 30°C, isolates produced pits on the CVP agar. Isolates were transferred onto NA and the incubation and transfer procedure was performed two additional times to obtain pure cultures. Suspensions of bacterial cells were injected into tomato and tobacco leaves to test for a hypersensitive or pathogenic reaction. Isolates produced collapsed necrotic tissue on tomato while no reaction was observed on tobacco. Tests for differentiating species and subspecies in the ‘carotovora’ group of Erwinia were conducted following the protocol of Dickey and Kelman (1). With known cultures of E. carotovora subsp. carotovora and E. chrysanthemi as controls, the isolate from tomato was determined to function as a facultative anaerobe, utilize asparagine as a sole source of carbon and nitrogen, and give positive reactions for pectate degradation, phosphatase, and growth at 37°C. Known cultures of E. carotovora subsp. carotovora, E. chrysanthemi, and the tomato isolate were grown on trypticase soy broth agar for 24 h at 28°C and their cellular fatty acids derivatized to fatty acid methyl esters (FAMEs). Statistical analyses of FAME profile data (MIDI Microbial Identification System, Newark, DE, version 3.60) identified the tomato isolate as Erwinia chrysanthemi. Pathogenicity was determined by inoculating 50-day-old tomato plants (cv. SunPride) with a suspension of E. chrysanthemi obtained from nutrient broth plates incubated at 24°C for 60 h. Three plants each were inoculated with the E. chrysanthemi identified from tomato, sterile water, and known cultures of E. chrysanthemi and E. carotovora subsp. carotovora by placing a drop at the junction of the petiole and stem and passing a sterile needle through the drop into the stem. Plants were maintained in a greenhouse. Dark, water-soaked cankers were observed on the stems of plants inoculated with E. chrysanthemi, including the tomato isolate and E. carotovora subsp. carotovora, after 7 days. No symptoms were observed on plants inoculated with sterile water. Reisolation of the pathogen and identification was performed with tissue from one of the symptomatic inoculated plants. Analyses of FAMEs confirmed E. chrysanthemi as the causal agent. This is the first report of E. chrysanthemi causing a vascular disease of field-grown tomato in Florida. Reference: (1) R. S. Dickey and A. Kelman. 1988. Pages 44–59 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. N. W. Schaad, ed. American Phytopathological Society, St. Paul, MN.

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Methods of plasma catecholamine measurement including radioimmunoassay

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1984.247.1.e4 ◽

1984 ◽

Vol 247 (1) ◽

pp. E4-E12 ◽

Cited By ~ 1

Author(s):

W. J. Raum

Keyword(s):

Reference Method ◽

Sample Volume ◽

Plasma Catecholamine ◽

Methyl Transferase ◽

Radioenzymatic Assay ◽

Specificity And Sensitivity ◽

Large Numbers ◽

Sensitivity Specificity ◽

Layer Chromatography ◽

Radioenzymatic Assays

Fluorometric and radioenzymatic assays are presently the most widely used techniques for the estimation of plasma, urine, and tissue catecholamines. The fluorometric assay lacks specificity and sensitivity. The radioenzymatic assay is significantly more sensitive and specific but is technically very complex, time consuming, and expensive. A newer methodology has been developed by modification of a 125I radioimmunoassay for metanephrine. The assay utilizes an antibody that specifically binds metanephrine. Plasma and urinary epinephrine and norepinephrine are detected by conversion to metanephrine with the enzymes catechol-O-methyl-transferase (COMT) and phenylethanolamine-N-methyltransferase (PNMT). The major advantages of the radioimmunoassay are the savings in cost and time. The radioenzymatic assay utilizes an expensive tritium-labeled compound, S-adenosylmethionine, and requires multiple organic solvent-extraction steps, thin-layer chromatography, and liquid scintillation counting. The radioimmunoassay requires only one extraction with alumina to aid in specificity and to concentrate the catecholamines. Sample detection is by gamma counting. The radioenzymatic assay is presently the reference method for catecholamines and is best suited for small numbers of samples where sample volume is limited and exquisite sensitivity is required. The radioimmunoassay is rapid, has sufficient sensitivity, specificity, and precision for most applications and is best applied to the analysis of large numbers of samples.

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Establishment of a New PNA-FISH Method for Aspergillus fumigatus Identification: First Insights for Future Use in Pulmonary Samples

Microorganisms ◽

10.3390/microorganisms8121950 ◽

2020 ◽

Vol 8 (12) ◽

pp. 1950

Author(s):

Laura Cerqueira ◽

Sara Moura ◽

Carina Almeida ◽

Maria João Vieira ◽

Nuno Filipe Azevedo

Keyword(s):

Aspergillus Fumigatus ◽

False Negative ◽

Clinical Samples ◽

Suitable Alternative ◽

Fish Method ◽

Negative Results ◽

Specificity And Sensitivity ◽

Pure Cultures ◽

Low Sensitivity ◽

Pna Fish

Aspergillus fumigatus is the main causative agent of Invasive Aspergillosis. This mold produces conidia that when inhaled by immunocompromized hosts can be deposited in the lungs and germinate, triggering disease. In this paper, the development of a method using peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) is described. The PNA-FISH probe was tested in several strains and a specificity and sensitivity of 100% was obtained. Detection of A. fumigatussensu stricto was then achieved in artificial sputum medium (ASM) pre-inoculated with 1 × 102 conidia·mL−1–1 × 103 conidia·mL−1, after a germination step of 24 h. The PNA-FISH method was evaluated in 24 clinical samples (10 sputum, 8 bronchoalveolar lavage (BAL), and 6 bronchial lavage (BL)) that were inoculated with 1 × 104 conidia·mL−1 in sputum; 1 × 103 conidia·mL−1 in BL and BAL, for 24 h. Despite a specificity of 100%, the sensitivity was 79%. This relatively low sensitivity can be explained by the fact that hyphae can yield “fungal ball“ clusters, hindering pipetting procedures and subsequent detection, leading to false negative results. Nonetheless, this study showed the potential of the PNA-FISH method for A. fumigatussensu stricto detection since it takes only 1 h 30 m to perform the procedure with a pre-enrichment step of 6 h (pure cultures) and 24 h (clinical samples), and might provide a suitable alternative to the existing methods for studies in pure cultures and in clinical settings.

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Effect of Enrichment Media and Sampling Protocol on Recovery of Listeria monocytogenes

Journal of Food Protection ◽

10.4315/0362-028x-53.6.505 ◽

1990 ◽

Vol 53 (6) ◽

pp. 505-507 ◽

Cited By ~ 6

Author(s):

J. S. BAILEY ◽

D. L. FLETCHER ◽

N. A. COX

Keyword(s):

Listeria Monocytogenes ◽

Pure Culture ◽

Sampling Protocol ◽

Enrichment Broth ◽

Secondary Enrichment ◽

Pure Cultures ◽

University Of Vermont

These studies examined the differences in recovery of Listeria monocytogenes from pure culture and in the populations of mixed aerobic microflora from chicken and Brie cheese incubated in University of Vermont (UVM) and Listeria enrichment broth (LEB) enrichment broths for different times and conditions. No significant differences were observed in levels of L. monocytogenes from pure cultures in UVM or LEB on any sampling day. No differences were observed in the levels of mixed microflora from Brie cheese in either UVM or LEB, but from chicken rinse the level of mixed flora competitors was significantly higher on all sampling days in LEB as compared to UVM. No differences were observed between a single enrichment in UVM or LEB for 2 d and a transfer to a secondary enrichment tube after 1 d. Overall, the level of mixed microflora capable of growing in enrichment broths was greater from chicken rinse than from Brie cheese. The ratio of L. monocytogenes to mixed microflora which survived the selective enrichments was most favorable for recovery of L. monocytogenes after 2 d of enrichment.

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Bolus Residue Scale: An Easy-to-Use and Reliable Videofluoroscopic Analysis Tool to Score Bolus Residue in Patients with Dysphagia

International Journal of Otolaryngology ◽

10.1155/2015/780197 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 11

Author(s):

Nathalie Rommel ◽

Charlotte Borgers ◽

Dirk Van Beckevoort ◽

Ann Goeleven ◽

Eddy Dejaeger ◽

...

Keyword(s):

Rating Scale ◽

Intraclass Correlation ◽

Correlation Coefficients ◽

Posterior Pharyngeal Wall ◽

High Specificity ◽

Analysis Tool ◽

Pharyngeal Wall ◽

Specificity And Sensitivity ◽

Low Sensitivity ◽

Sensitivity Specificity

Background. We aimed to validate an easy-to-use videofluoroscopic analysis tool, the bolus residue scale (BRS), for detection and classification of pharyngeal retention in the valleculae, piriform sinuses, and/or the posterior pharyngeal wall.Methods. 50 randomly selected videofluoroscopic images of 10 mL swallows (recorded in 18 dysphagia patients and 8 controls) were analyzed by 4 experts and 6 nonexpert observers. A score from 1 to 6 was assigned according to the number of structures affected by residue. Inter- and intrarater reliabilities were assessed by calculation of intraclass correlation coefficients (ICCs) for expert and nonexpert observers. Sensitivity, specificity, and interrater agreement were analyzed for different BRS levels.Results. Intrarater reproducibility was almost perfect for experts (mean ICC 0.972) and ranged from substantial to almost perfect for nonexperts (mean ICC 0.835). Interjudge agreement of the experts ranged from substantial to almost perfect (mean ICC 0.780), but interrater reliability of nonexperts ranged from substantial to good (mean 0.719). BRS shows for experts a high specificity and sensitivity and for nonexperts a low sensitivity and high specificity.Conclusions. The BRS is a simple, easy-to-carry-out, and accessible rating scale to locate pharyngeal retention on videofluoroscopic images with a good specificity and reproducibility for observers of different expertise levels.

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Taxonomic Studies on Australian Isolates of Stemphylium spp. And Associated Teleomorphs

Australian Journal of Botany ◽

10.1071/bt9860281 ◽

1986 ◽

Vol 34 (3) ◽

pp. 281 ◽

Cited By ~ 3

Author(s):

JAG Irwin ◽

R Dill-Macky ◽

M Stirling

Keyword(s):

Medicago Sativa ◽

Pure Culture ◽

Herbarium Specimens ◽

Simmondsia Chinensis ◽

Pure Cultures ◽

Taxonomic Studies ◽

Ascospore Morphology

Taxonomic studies on monosporic pure cultures of Australian isolates of Stemphylium revealed the presence of three species, namely S. botryosum, S. globuliferum and S. vesicarium. A group intermedi- ate between S. botryosum and S. vesicarium but more closely resembling S. vesicarium was recognised. The circumscription of S. vesicarium was broadened to accommodate this taxon which was found on Medicago sativa, M. rugosa and Simmondsia chinensis. Studies on herbarium specimens deposited as S. botryosum on M. sativa from a number of overseas locations indicated that in all cases the pathogen fitted our concept of S. vesicarium. Teleomorphs were induced in pure culture for each of the above-mentioned anamorphs, and in all instances the teleomorph was identified as Pleospora herbarum. The ascospore morphology was similar for all isolates, and the shape of the ends of the spore and the number of longitudinal septa were variable characters even within an isolate. However, the teleomorphs could be separated into two distinct groups on the basis of ascal length.

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