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)

On relaxed LMI-based designs for fuzzy regulators and fuzzy observers

Relaxed conditions for stability of nonlinear, continuous and discrete-time systems given by fuzzy models are presented. A theoretical analysis shows that the proposed methods provide better or at least the same results of the methods presented in the literature. Numerical results exemplify this fact. These results are also used for fuzzy regulators and observers designs. The nonlinear systems are represented by fuzzy models proposed by Takagi and Sugeno. The stability analysis and the design of controllers are described by linear matrix inequalities, that can be solved efficiently using convex programming techniques. The specification of the decay rate, constrains on control input and output are also discussed.

Robust state-derivative pole placement LMI-based designs for linear systems

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Robust State-Derivative Feedback LMI-Based Designs for Linear Descriptor Systems

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Accuracy of six minute walk test, stair test and spirometry using maximal oxygen uptake as gold standard

OBJETIVO: Determinar a acurácia das variáveis: tempo de escada (tTE), potência de escada (PTE), teste de caminhada (TC6) e volume expiratório forçado (VEF1) utilizando o consumo máximo de oxigênio (VO2máx) como padrão-ouro. MÉTODOS: Os testes foram realizados em 51 pacientes. O VEF1 foi obtido através da espirometria. O TC6 foi realizado em corredor plano de 120m. O TE foi realizado em escada de 6 lances obtendo-se tTE e PTE. O VO2máx foi obtido por ergoespirometria, utilizando o protocolo de Balke. Foram calculados a correlação linear de Pearson (r) e os valores de p, entre VO2máx e variáveis. Para o cálculo da acurácia, foram obtidos os pontos de corte, através da curva característica operacional (ROC). A estatística Kappa (k) foi utilizada para cálculo da concordância. RESULTADOS: Obteve-se as acurácias: tTE - 86%, TC6 - 80%, PTE - 71%, VEF1(L) - 67%, VEF1% - 63%. Para o tTE e TC6 combinados em paralelo, obteve-se sensibilidade de 93,5% e em série, especificidade de 96,4%. CONCLUSÃO: O tTE foi a variável que apresentou a melhor acurácia. Quando combinados o tTE e TC6 podem ter especificidade e sensibilidade próxima de 100%. Estes testes deveriam ser mais usados rotineiramente, especialmente quando a ergoespirometria para a medida de VO2máx não é disponível.

Comparison between actual and predicted postoperative stair-climbing test, walk test and spirometric values in patients undergoing lung resection

OBJETIVO: Verificar se os testes: Volume Expiratório Forçado no 1º segundo (VEF1), Teste de Caminhada de 6 minutos (TC6) e Teste de Escada (TE) se alteram proporcionalmente ao pulmão funcionante ressecado. MÉTODOS: Foram incluídos pacientes candidatos a toracotomia para ressecção pulmonar. No pré-operatório (pré) e no mínimo três meses após a cirurgia (pós), realizaram espirometria, TC6 e TE. O TE foi realizado em escada com 12,16m de altura. O tempo para subir todos os degraus o mais rápido possível foi chamado tempo de escada (tTE). Os cálculos dos valores dos testes preditos para o pós-operatório (ppo) foram realizados conforme o número de segmentos funcionantes perdidos. Os valores pré, ppo e pós foram comparados entre si para cada teste. Estatística: foi utilizada a análise de variância para medidas repetidas (ANOVA), com significância de 5%. RESULTADOS: Foram estudados 40 pacientes. A ressecção pulmonar variou desde o ganho de dois segmentos funcionantes até a perda de 9. Os valores pré, ppo e pós foram respectivamente: VEF1 -pré = 2,6±0,8L, ppo = 2,3±0,8L, pós = 2,3±0,8L (VEF1pré > VEF1ppo = VEF1pós), TC6-pré = 604±63m, ppo = 529±103m, pós = 599±74m (TC6pré = TC6pós > TC6ppo), tTE-pré = 32,9±7,6s, ppo = 37,8±12,1s, pós = 33,7±8,5s (tTEpré = tTEpós < tTEppo). CONCLUSÃO: Nas ressecções pulmonares, este grupo de pacientes perdeu função pulmonar medida através da espirometria, mas não perdeu a capacidade de exercício, medida através dos testes de escada e caminhada.

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)

Comparative Analysis of 2-Flap Designs for Extraction of Mandibular Third Molar

Objective: The objective of the study was to analyze 2-flap designs for surgical extraction of third molar, evaluating the periodontal status of the second lower molar.Study Design: Forty-five lower third molars were extracted from 24 patients. In 23 teeth, a vertical incision to the mandibular ramus was used (technique A), whereas 22 teeth were submitted to classic L-shaped flap (technique B) with controls at 60 and 90 days postoperatively.Results: Pearson correlation coefficient analysis showed a significant correlation only between immediate preoperative probing depth variables from techniques A and B in the studied surfaces. Statistical significances in the preoperative (vestibular) and postoperative day 60 (distovestibular and vestibular) were noted. In contrast, Student t-test showed no statistical difference in probing depths between preoperative and postoperative values, as well as no statistically significant difference regarding the type of incision alone.Conclusions: Technique A allowed a less traumatic surgery, guaranteeing a more comfortable postoperative period.

Multistratum response surface designs

Response surface designs are usually described as if the treatments have been completely randomized to the experimental units. However, in practice there is often a structure to the units, implying the need for blocking. If, in addition, some factors are more difficult to vary between units than others, a multistratum structure arises naturally. We present a general strategy for constructing response surface designs in multistratum unit structures. Designs are constructed stratum by stratum, starting in the highest stratum. In each stratum a prespecified treatment set for the factors applied in that stratum is arranged to be nearly orthogonal to the units in the higher strata, allowing-for all the effects that have to be estimated. Three examples are given to show the applicability of the method and are also used to check the relationship of the final design to the choice of treatment set. Finally, some practical considerations in randomization are discussed.

An algorithm for arranging response surface designs in small blocks

We consider the problem of blocking response surface designs when the block sizes are prespecified to control variation efficiently and the treatment set is chosen independently of the block structure. We show how the loss of information due to blocking is related to scores defined by Mead and present an interchange algorithm based on scores to improve a given blocked design. Examples illustrating the performance of the algorithm are given and some comparisons with other designs are made. (C) 2000 Elsevier B.V. B.V. All rights reserved.