Infectious bronchitis virus: detection and vaccine Strain differentiation by semi-nested RT-PCR

A semi-nested reverse transcription-polymerase chain reaction (Semi-N-RT-PCR) was developed and used to detect the S glycoprotein gene of infectious bronchitis virus (IBV) strains and to discriminate H120 vaccine strain from other strains. Viral RNA was extracted from the allantoic fluid of chicken embryos and from tissues of chickens experimentally infected with different strains of IBV. Amplification and identification of the viral RNA was performed using two sets of primers complementary to a region of the S glycoprotein gene in the Semi-N-RT-PCR assay. The pair of primers used in the first PCR consisted of universal oligonucleotides flanking a more variable region of S1-S2 gene. The second primer pair was used in the Semi-N-RT-PCR and was comprised of one of the primers from the first universal pair together with either another universal internal oligolucleotide or a oligonucleotide sequence specific for the H120 strain of IBV. The universal primers detected all reference IBV strains and field isolates tested herein. The Semi-N-RT-PCR had high sensitivity and specificity, and was able to differentiate the H120 vaccine strain from other reference IBV strains; including M41 strain. All tissue samples collected from chickens experimentally infected with H120 or M41 strains were positive in the semi-nested RT-PCR using universal primers, while only the H120-infected tissue samples were amplified by the set of primers containing the H120-oligonucleotide. In conclusion, the ability of Semi-N-RT-PCR to detect distinct IBV strains and preliminarily discriminate the vaccine strain (H120) closes a diagnostic gap and offers the opportunity to use comprehensive PCR procedures for the IBV diagnosis.

In vitro culture, PCR , and nested PCR for the detection of Theileria equi in horses submitted to exercise

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detection of bovine herpesvirus 5 (BoHV-5) in formalin-fixed, paraffin-embedded bovine brain by nested PCR in Colombian cattle

Fifteen cases of viral meningoencephalitis in Colombian cattle were tested by nested PCR analysis for the detection of bovine herpesvirus 5 (BoHV-5). All fatal cases had shown severe neurological signs and had occurred following natural outbreaks of the disease. The neurological infection was histologically characterized by mild to moderate inflammatory changes in the brain and cerebellum, including meningitis, mononuclear perivascular cuffing, gliosis, haemorrhage, and the presence of Gitter cells (macrophages) accompanying large areas of malacia. No intranuclear inclusion bodies were seen in any of the cases. Results from BoHV-5 molecular extraction analyses showed there were five positive cases thus confirming the presence of the virus in Colombia.

A nested-PCR assay for detection of Xylella fastidiosa in citrus plants and sharpshooter leafhoppers

Aims: Detection of Xylella fastidiosa in citrus plants and insect vectors.Methods and Results: Chelex 100 resin matrix was successfully standardized allowing a fast DNA extraction of X. fastidiosa. An amplicon of 500 bp was observed in samples of citrus leaf and citrus xylem extract, with and without symptoms of citrus variegated chlorosis, using PCR with a specific primer set indicating the presence of X. fastidiosa. The addition of insoluble acid-washed polyvinylpyrrolidone (PVPP) prior to DNA extraction of insect samples using Chelex 100 resin together with nested-PCR permitted the detection of X. fastidiosa within sharpshooter heads with great sensitivity. It was possible to detect up to two bacteria per reaction. From 250 sharpshooter samples comprising four species (Dilobopterus costalimai, Oncometopia facialis, Bucephalogonia xanthopis and Acrogonia sp.), 87 individuals showed positive results for X. fastidiosa in a nested-PCR assay.Conclusions: the use of Chelex 100 resin allowed a fast and efficient DNA extraction to be used in the detection of X. fastidiosa in citrus plants and insect vectors by PCR and nested-PCR assays, respectively.Significance and Impact of the study: the employment of efficient and sensitive methods to detect X. fastidiosa in citrus plants and insect vectors will greatly assist epidemiological studies.

Nested liana-tree network in three distinct neotropical vegetation formations

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Brazilian Students and Working Capital: An Analysis of Their Decisions When Using Virtual Market Business Games

This study aims to demonstrate that data from business games can be an important resource for improving efficiency and effectiveness of learning. The proposal presented here was developed from preliminary studies of data from Virtual Market games that pointed the possibility of identifying gaps in learning by analyzing the decisions of students. This proposal helps students to refine their learning processes and equips tutors with strategies for teaching and student assessment. The proposal also complements the group discussion and/or debriefing, which are widely used to enhance learning mediated by games. However, from a management perspective the model has the potential to be erroneous and miss opportunities, which cannot be detected because of the dependence on the characteristics of the individual, such as ability to communicate and work together. To illustrate the proposed technique, data sets from two business games were analyzed with the focus on managing working capital and it was found that students had difficulties managing this task. Similar trends were observed in all categories of students in the study-undergraduate, postgraduate and specialization. This discovery led us to the analysis of data for decisions made in the performance of the games, and it was determined that indicators could be developed that were capable of indentifying inconsistencies in the decisions. It was decided to apply some basic concepts of the finance management, such as management of the operational and non-operational expenditures, as well as production management concepts, such as the use of the production capacity. By analyzing the data from the Virtual Market games using the indicator concept, it was possible to detect the lack of domain knowledge of the students. Therefore, these indicators can be used to analyze the decisions of the players and guide them during the game, increasing their effectiveness and efficiency. As these indicators were developed from specific content, they can also be used to develop teaching materials to support learning. Viewed in this light, the proposal adds new possibilities for using business games in learning. In addition to the intrinsic learning that is achieved through playing the games, they also assist in driving the learning process. This study considers the applications and the methodology used.

Typing class I HLA-A gene using a nested PCR-RFLP procedure

In order to detect several new HLA-A class I alleles that have been described since 1998, the original PCR-RFLP method developed to identify the 78 alleles recognized at that time at high resolution level was adapted by us for low and medium resolution levels using a nested PCR-RFLP approach. The results obtained from blood samples of 23 subjects using both the PCR-RFLP method and a commercial kit (MicroSSP1A®, One Lambda Inc.) showed an agreement higher than 95%. The PCR-RFLP adapted method was effective in low and medium resolution histocompatibility evaluations.

Species-specific nested PCR as a diagnostic tool for Brucella ovis infection in rams

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparison of visual inspection, radiographic examination, laser fluorescence and their combinations on treatment decisions for occlusal surfaces

Purpose: To compare visual inspection (VI), radiographic examination (RX) and the laser fluorescence device DIAGNOdent (L), as well as their combinations in vitro regarding treatment decisions for occlusal surfaces. Methods: 72 extracted human permanent teeth (molars and premolars) were used. Treatment decisions were recorded by three calibrated examiners, and the options available were fissure sealant and conservative restoration. For validation of treatment decisions, the teeth were sectioned and examined in a stereomicroscope. Thereafter, dental slices were scanned and the images were edited to facilitate classification of existing carious lesions. Intra and inter-examiner reproducibility for the determination of treatment plans were calculated using Cohen's kappa test (95%-CI). Sensitivity, specificity, positive and negative predictive values, and the area under the ROC curve were also calculated. Results: VI and L provided on average the greatest intra- and inter-examiner reproducibility, respectively. Although the combination of diagnostic methods may decrease both intra- and inter examiners reproducibility, combination of VI, L and RX resulted in the greatest sensitivity, being statistically superior to RX and L. There was more inter-examiner agreement for the option of restorative treatment, while the use of sealants as a treatment option yielded the lowest values. Negative predictive values were numerically inferior to positive predictive values, indicating that the examiners preferred not to restore a carious tooth than to proceed operatively in an intact tooth. The combination of the three methods studied showed the best results in determining treatment plans for occlusal surfaces, when compared to the other types of exams. on the other hand, radiographic examination and laser fluorescence were less efficient when used alone.

Advantages of using nested collision induced dissociation/post-source decay with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: sequencing of novel peptides from wasp venom

We have examined the applicability of the 'nested' collision induced dissociation/post-source decay (CID/PSD) method to the sequencing of novel peptides from solitary wasps which have neurotoxic venom for paralyzing other insects. The CID/PSD spectrum of a ladder peptide derived from an exopeptidase digest was compared with that of the intact peptide. The mass peaks observed only in the CID/PSD spectrum of a ladder peptide were extracted as C-terminal fragment ions. Assignment of C-terminal fragment ions enabled calculation of N-terminal fragment masses, leading to differentiation between N-terminal fragment ions and internal fragment ions. This methodology allowed rapid and sensitive identification by removing ambiguity in the assignment of the fragment ions, and proved useful for sequencing unknown peptides, in particular those available as natural products with a limited supply. Copyright (C) 2000 John Wiley & Sons, Ltd.

When decisions on homologous structures cause ambiguous taxa relationships: the Neomorphinae (Aves, Cuculidae) example

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sequencing wasp venom peptides by endopeptidase digestion and nested collision-induced dissociation/post-source decay methods

A method incorporating nested collision-induced dissociation/post-source decay (CID/PSD) combined with endopeptidase digestion is described as an approach to determine the sequence of N-terminally modified peptides. The information from immonium and related ions observed in the CID/PSD spectrum was used for the selection of a suitable endopeptidase for the digestion of peptides. Rapid and reliable assignment of peptide sequence was performed by the comparison of CID/PSD spectra of both intact and endopeptidese-digested peptide fragments, since the assignments of the observed fragment ions to either N- or C-terminal ions can thus be carried out unambiguously. This nested CID/PSD method was applied to the sequence determination of two peptides from the solitary wasps Anoplius samariensis and Batozonellus maculifrons (pompilid wasps), which could not be sequenced by the Edman method due to N-terminal modification. Copyright (C) 2002 John Wiley Sons, Ltd.

In Vitro Evaluation of ICDAS and Radiographic Examination of Occlusal Surfaces and Their Association With Treatment Decisions

This in vitro study evaluated the performance of visual (International Caries Detection and Assessment System [ICDAS]) and radiographic (bitewing [BW]) examinations for occlusal caries detection and their associations with treatment decision (TD). Permanent teeth (n=104) with occlusal surfaces varying from sound to cavitated were selected. Sites were identified from 10x occlusal surface photographs. Standardized bitewing (BW) radiographs were taken. Four dentists with at least five years of experience scored all teeth twice (one-week interval) for ICDAS (0-6), BW (0=sound, 1=caries restricted to enamel, 2=caries in outer third dentin, 3=caries in inner third dentin), and TD (0=no treatment, 1=sealant, 2=microabrasion and sealant, 3=round bur sealant, 4a=resin, 4b=amalgam). Histological validation was performed by observation under a light microscope, with lesions classified on a five-point scale. Intraexaminer and inter-examiner repeatability were assessed using two-way tables and intraclass correlation coefficients (ICCs). Comparisons between percentage correct, specificity, sensitivity, and area under the receiver-operating characteristic (ROC) curve were performed using bootstrap analyses. ICCs for intraexaminer and interexaminer repeatability indicated good repeatability for each examiner, ranging from 0.78 to 0.88, and among examiners, ranging from 0.74 to 0.81. Correlation between ICDAS and TD was 0.85 and between BW and TD was 0.78. Correlation between the methods and histological scores was moderate (0.63 for ICDAS and 0.61 for BW). The area under the ROC curve was significantly greater for ICDAS than for BW (p<0.0001). ICDAS had significantly lower specificity than BW did (p=0.0269, 79% vs 94%); however, sensitivity was much higher for ICDAS than for BW (p<0.0001, 83% vs 44%). Data from this investigation suggested that the visual examination (ICDAS) showed better performance than radiographic examination for occlusal caries detection. The ICDAS was strongly associated with TD. Although the correlation between the ICDAS and BW was lower, it is still valuable in the clinical decision-making process.

Strategies to model scheduling decisions to plan the soft drink production process

In this paper we present a mixed integer model that integrates lot sizing and lot scheduling decisions for the production planning of a soft drink company. The main contribution of the paper is to present a model that differ from others in the literature for the constraints related to the scheduling decisions. The proposed strategy is compared to other strategies presented in the literature.

Model of a game for improving integrated decisions in production management

This paper presents the results obtained with a business game whose model represents the decision making process related to two moments at an industrial company. The first refers to the project of the industrial plant, and the second to its management. The game model was conceived so the player's first decision would establish capacity and other parameters such as quantities of each product to produce, marketing expenses, research and development, quality, advertising, salaries, if purchases will be made in installments or in cash, if there will be credit sales and how many installments will be allowed and the number of workers in the assembly area. An experiment was conducted with employees of a Brazilian company. Data obtained indicate that the players have lack of contents, especially in finances. Although these results cannot be generalized, they confirm prior results with undergraduate and graduate students and they indicate the need for reinforcement in this undergraduate area. © 2012 Springer-Verlag.