PrimerSNP: a web tool for whole-genome selection of allele-specific and common primers of phylogenetically-related bacterial genomic sequences

Background: The increasing number of genomic sequences of bacteria makes it possible to select unique SNPs of a particular strain/species at the whole genome level and thus design specific primers based on the SNPs. The high similarity of genomic sequences among phylogenetically-related bacteria requires the identification of the few loci in the genome that can serve as unique markers for strain differentiation. PrimerSNP attempts to identify reliable strain-specific markers, on which specific primers are designed for pathogen detection purpose.Results: PrimerSNP is an online tool to design primers based on strain specific SNPs for multiple strains/species of microorganisms at the whole genome level. The allele-specific primers could distinguish query sequences of one strain from other homologous sequences by standard PCR reaction. Additionally, PrimerSNP provides a feature for designing common primers that can amplify all the homologous sequences of multiple strains/species of microorganisms. PrimerSNP is freely available at http://cropdisease.ars.usda.gov/similar to primer.Conclusion: PrimerSNP is a high-throughput specific primer generation tool for the differentiation of phylogenetically-related strains/species. Experimental validation showed that this software had a successful prediction rate of 80.4 - 100% for strain specific primer design.

Evaluation of DNA polymorphisms amplified by arbitrary primers (RAPD) as genetically associated elements to differentiate virulent and non-virulent Paracoccidioides brasiliensis isolates

Randomly amplified polymorphic DNA (RAPD) analysis of 35 Paracoccidioides brasiliensis isolates was carried out to evaluate the correlation of RAPD profiles with the virulence degree or the type of the clinical manifestations of human paracoccidioidomycosis. The dendrogram presented two main groups sharing 64% genetic similarity. Group A included two isolates from patients with chronic paracoccidioidomycosis; group B comprised the following isolates showing 65% similarity: two non-virulent, six attenuated, five virulent, eight from patients with chronic paracoccidioidomycosis and two from patients with acute paracoccidioidomycosis. The virulent Pb18 isolate and six attenuated or non-virulent samples derived from it were genetically indistinguishable (100% of similarity). Thus, in our study, RAPD patterns could not discriminate among 35 P. brasiliensis isolates according to their differences either in the degree of virulence or in the type of the clinical manifestation of this fungal infection. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Aplicação da técnica de condicionamento total ou de primers autocondicionantes em dentes decíduos após abrasão a ar

Since the use of air abrasion has grown in pediatric dentistry, the aim of this study was to evaluate, by means of shear bond strength testing, the need to use the total etching technique or self-etching primers on dentin of primary teeth after air abrasion. Twenty-five exfoliated primary molars had their occlusal dentin exposed by trimming and polishing. Specimens were treated by: Air abrasion + Scotchbond MultiPurpose adhesive (G1); 37% phosphoric acid + Scotchbond MP adhesive (G2); Clearfil SE (G3); Air abrasion ( 37% phosphoric acid + Scotchbond MP adhesive (G4); Air abrasion + Clearfil SE (G5). On the treated surface, a cylinder of 2 mm by 6 mm was made using a composite resin (Z100). Duncan's test showed that: (G2 = G3 = G5) > (G1 = G4). The use of a selfetching primer on air abraded dentin is recommended to obtain higher bond strengths.

Optimal clone identifier for genomic shotgun libraries: OC Identifier tool

In DNA microarray experiments, the gene fragments that are spotted on the slides are usually obtained by the synthesis of specific oligonucleotides that are able to amplify genes through PCR. Shotgun library sequences are an alternative to synthesis of primers for the study of each gene in the genome. The possibility of putting thousands of gene sequences into a single slide allows the use of shotgun clones in order to proceed with microarray analysis without a completely sequenced genome. We developed an OC Identifier tool (optimal clone identifier for genomic shotgun libraries) for the identification of unique genes in shotgun libraries based on a partially sequenced genome; this allows simultaneous use of clones in projects such as transcriptome and phylogeny studies, using comparative genomic hybridization and genome assembly. The OC Identifier tool allows comparative genome analysis, biological databases, query language in relational databases, and provides bioinformatics tools to identify clones that contain unique genes as alternatives to primer synthesis. The OC Identifier allows analysis of clones during the sequencing phase, making it possible to select genes of interest for construction of a DNA microarray. ©FUNPEC-RP.

Influence of acid-etching and ceramic primers on the repair of a glass ceramic

The objective of this study was to evaluate the influence of different primers on the microtensile bond strength (μT BS) between a feldspathic ceramic and two composites. Forty blocks (6.0 × 6.0 × 5.0 mm 3) were prepared from Vita Mark II . After polishing, they were randomly divided into 10 groups according to the surface treatment: Group 1, hydrofluoric acid 10% (HF) + silane; Group 2, CoJet + silane; Group 3, HF + Metal/Zirconia Primer; Group 4, HF + Clearfil Primer; Group 5, HF + Alloy Primer; Group 6, HF + V-Primer; Group 7, Metal/Zirconia Primer; Group 8, Clearfil Primer; Group 9, Alloy Primer; Group 10, V-Primer. After each surface treatment, an adhesive was applied and one of two composite resins was incrementally built up. The sticks obtained from each block (bonded area: 1.0 mm2 ± 0.2 mm) were stored in distilled water at 37°C for 30 days and submitted to thermocycling (7,000 cycles; 5°C/55°C ± 1°C). The μT BS test was carried out using a universal testing machine (1.0 mm/min). Data were analyzed using ANOVA and a Tukey test (α = 0.05). The surface treatments significantly affected the results (P < 0.05); no difference was observed between the composites (P > 0.05). The bond strength means (MPa) were as follows: Group 1a = 29.6; Group 1b = 33.7; Group 2a = 28.9; Group 2b = 27.1; Group 3a = 13.8; Group 3b = 14.9; Group 4a = 18.6; Group 4b = 19.4; Group 5a = 15.3; Group 5b = 16.5; Group 6a = 11; Group 6b = 18; Groups 7a to 10b = 0. While the use of primers alone was not sufficient for adequate bond strengths to feldspathic ceramic, HF etching followed by any silane delivered higher bond strength.

Epidemiologia da infecção genital pelo Papilomavírus humano (HPV) em população feminina geral e população carcerária

A infecção genital pelo Papilomavírus humano (HPV) é considerada uma das doenças sexualmente transmissíveis (DSTs) mais comum, representando um importante problema na Saúde Pública, além de estar diretamente relacionado à promoção do câncer de colo uterino. Este estudo teve o intuito de investigar os aspectos epidemiológicos da infecção genital pelo HPV em dois grupos distintos: mulheres de população geral e mulheres encarceradas. Para tanto foi conduzido um estudo transversal analítico com 423 mulheres a partir dos 18 anos que se submeteram ao exame preventivo do câncer do colo uterino, sendo 233 mulheres da população geral oriundas de uma unidade básica de saúde da cidade de Belém do Pará e 190 provenientes do Centro de Reeducação Feminino em Ananindeua no mesmo Estado, no período de janeiro de 2008 a março de 2010. Amostras da cérvice uterina foram coletadas para a realização da colpocitologia convencional e para a detecção do DNA do HPV através da reação em cadeia da polimerase (PCR) mediada pelos oligonucleotídeos iniciadores universais MY9/11. Todas as mulheres responderam a um formulário clínico e epidemiológico. Entre as 423 mulheres analisadas, a prevalência geral de infecção genital pelo HPV foi de 13,0% com variação entre 15,0% para a amostra geral e 10,5% para a carcerária. A faixa etária mais acometida foi a de 13 a 25 anos (19%) na amostra geral; e em mulheres com 45 anos ou mais (21,1%), nas carcerárias. Anormalidades Colpocitológicas, situação conjugal, número de parceiros sexuais novos, o uso de anticoncepcionais orais, história de DST e de sintomas genitais, além de tabagismo atual, foram fatores que se mostraram associados à infecção genital pelo HPV de maneira diferenciada entre amostras da população geral e carcerária.

The potential of novel primers and universal adhesives to bond to zirconia

Objectives: To investigate the adhesive potential of novel zirconia primers and universal adhesives to surface-treated zirconia substrates.Methods: Zirconia bars were manufactured (3.0 mm x 3.0 mm x 9.0 mm) and treated as follows: no treatment (C); air abrasion with 35 mu m alumina particles (S); air abrasion with 30 mu m silica particles using one of two systems (Rocatec or SilJet) and; glazing (G). Groups C and S were subsequentially treated with one of the following primers or adhesives: ZP (Z-Prime Plus), AZ (AZ Primer); MP (Monobond Plus); SU (ScotchBond Universal) and; EA (an Experimental Adhesive). Groups Rocatec and SilJet were silanized prior to cementation. Samples form group G were further etched and silanized. Bars were cemented (Multilink) onto bars of a silicate-based ceramic (3.0 mm x 3.0 mm x 9.0 mm) at 90 degrees angle, thermocycled (2.500 cycles, 5-55 degrees C, 30 s dwell time), and tested in tensile strength test. Failure analysis was performed on fractured specimens to measure the bonding area and crack origin.Results: Specimens from group C did not survive thermocycling, while CMP, CSU and CEA groups survived thermocycling but rendered low values of bond strength. All primers presented a better bond performance after air abrasion with Al2O3 particles. SilJet was similar to Rocatec, both presenting the best bond strength results, along with SMP, SSU and CEA. G promoted intermediate bond strength values. Failure mode was predominately adhesive on zirconia surface combined to cohesive of the luting agent.Conclusions: Universal adhesives (MP, SU, EA) may be a considerable alternative for bonding to zirconia, but air abrasion is still previously required. Air abrasion with silica particles followed by silane application also presented high bond strength values. (C) 2013 Elsevier Ltd. All rights reserved.

Estudo comparativo in vitro da resistência ao cisalhamento de sistemas adesivos em dentina humana: uso de hipoclorito de sódio em sistemas convencionais ou primers acidificados

Pós-graduação em Odontologia Restauradora - ICT

Avaliação da eficácia de primers para metal na resistência ao cisalhamento da união entre cimentos resinosos e metais não nobres. Efeito do armazenamento em água

O sucesso das restaurações metálicas indiretas depende, dentre outros fatores, de uma união eficaz entre a estrutura metálica e o cimento dentário empregados. Sabe-se que os primers para metal atuam com comprovada eficácia na resistência adesiva dos cimentos resinosos às ligas nobres. Entretanto, pouco foi estudado sobre o efeito de tais materiais em ligas não nobres. O propósito deste estudo foi avaliar a eficácia de primers para metal na resistência ao cisalhamento da união entre cimentos resinosos e metais não nobres. Discos (9 mm de diâmetro e 3 mm de altura) foram fundidos em liga de NiCr (n=80) e em titânio comercialmente puro (Ti c.p.) (n=80) e foram incluídos em anel de PVC com resina acrílica quimicamente ativada. As superfícies dos discos foram regularizadas com lixas de carbeto de silício de granulação 320, 400 e 600 e jateadas com partículas de óxido de alumínio de 50 µm. Espécimes de cada metal foram divididos em quatro grupos (n=20). Uma matriz metálica bi-partida (5 mm de diâmetro interno e 2 mm de altura) foi posicionada na superfície do espécime. As áreas adesivas receberam um dos seguintes tratamentos: 1) Panavia F; 2) Alloy Primer e Panavia F; 3) Bistite DC e 4) Metaltite e Bistite DC. Para evitar a exposição dos cimentos à luz, estes foram espatulados e inseridos na matriz dentro de uma câmara de revelação radiográfica. Quarenta minutos após a confecção, os espécimes foram armazenados em água destilada a 37ºC por 24 horas e então termociclados (1.000 ciclos, 5ºC e 55ºC, 30 segundos cada banho). Após a termociclagem, os espécimes foram armazenados novamente nas mesmas condições descritas anteriormente por um período de 24 horas (n=10) ou de 6 meses (n=10) antes do ensaio de cisalhamento em uma máquina de ensaios mecânicos (Material Test System 810).

Can Application of Universal Primers Alone Be a Substitute for Airborne-Particle Abrasion to Improve Adhesion of Resin Cement to Zirconia?

Purpose: To determine whether universal primers alone can deliver similar levels of adhesion of resin cement to zirconia ceramic when compared to their application in conjunction with airborne-particle abrasion.Materials and Methods: Sintered zirconia blocks (N = 160) (Lava, 3M ESPE), (5.25 x 5.25 x 3 mm(3)) were embedded in acrylic resin, polished, and randomly distributed into 16 groups (n = 10 per group), according to the factors "universal primer" (8 levels) and "air-particle abrasion" (2 levels): 1. ctr: control, without application of a universal primer; 2. AP: Alloy Primer; 3. MP: Monobond Plus; 4. MZP: Metal Zirconia Primer; 5. MZ: MZ Primer; 6. Sg: Signum Zirconia Bond; 7. SbU: Singlebond Universal; 8. ZP: Z Prime Plus. The universal primers were also used after air abrasion (A) of zirconia to form the following 8 groups: Ctr-A, AP-A, MP-A, MZP-A, MZ-A, Sg-A, SbU-A, and ZP-A. After ultrasonic cleaning, air abrasion was performed using Al2O3 particles (110 mu m, 2.5 bar, 20 s at 10 mm) in a chairside air-abrasion device. After ultrasonic cleaning again, universal primers were applied according to each manufacturer's recommendation. The resin cement (RelyX ARC, 3M ESPE) was built up incrementally and photo-polymerized on the zirconia surface using a silicone mold (empty set = 3.5, height = 3 mm). All specimens were stored in distilled water (60 days at 37 degrees C) and then subjected to shear bond strength testing (SBS) in a universal testing machine (1 mm/min). On a separate set of zirconia specimens, contact angle measurements were made using the sessile drop technique with a goniometer after the application of universal primers on control and air-abraded zirconia surfaces. Data (MPa) were analyzed using one-way ANOVA, Tukey's test, and Student's t-test (alpha = 0.05).Results: When universal primers were used alone, SbU presented significantly higher mean SBS (19.5 +/- 5.8) that did the other primers (0 to 9.9 +/- 6.6) (p = 0.001). When air abraded, the groups AP-A (14.1 +/- 6.1), MP-A (15.9 +/- 5.4), ZP-A (16.9 +/- 7.3), SG-A (19.1 +/- 2.1), SbU-A (12 +/- 1.5) showed significant differences (p = 0.03). Adhesive performance of all universal primers was enhanced after air abrasion, with the exception of the SbU and MZ primers. After air abrasion, contact angle measurements were lower for the each primer (without air abrasion: 28.9 to 83.9; with air abrasion: 27.1 to 63.0), except for MZP.Conclusion: Air abrasion with 110 mu m Al2O3 followed by universal primer application increased the bond strength of tested resin cement to zirconia, with the exception of SbU and MZ.

Hemoculture and polymerase chain reaction using primers TCZ1/TCZ2 for the diagnosis of canine and feline trypanosomiasis

Introduction. American trypanosomiasis, also known as Chagas disease, is a zoonosis caused by Trypanosoma cruzi (T. cruzi). Dogs and cats participate actively in this parasite's transmission cycle. This study aimed at evaluating the occurrence of T. cruzi in dogs and cats from Botucatu, SP, Brazil, as well as at evaluating the technique of hemoculture in LIT (liver infusion tryptose) medium by polymerase chain reaction (PCR). Methods. Blood samples were collected from 50 dogs and 50 cats in Botucatu-SP, Brazil. For hemoculture, the samples were inoculated in LIT medium, and readings were performed for four months. Upon completion of such period, all the hemocultures were processed for parasitic DNA extraction. The PCR reactions were performed by using primers TCZ1/TCZ2. Results. Ten dogs and ten cats (20%) were positive to PCR, and four dogs and three cats (7%) were positive to hemoculture. Only in a one cat sample (1%) there was confirmation of positive hemoculture by PCR for T. cruzi. Conclusions. Results showed that PCR was a suitable tool for the confirmation of the parasite detection in hemoculture samples, and that dogs and cats from Botucatu, SP, Brazil, are maintaining the role of household reservoirs of T. cruzi, which reinforces the need for constant epidemiologic surveillance for this zoonosis.

Biocompatibilidade de primers e adesivo utilizados na confecção de próteses maxilofaciais implantorretidas: análise in vitro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Application of Microsatellite Primers Developed for Polistes in the Independent-Founding Wasp Polists satan Bequaert (Hymenoptera: Vespidae)

Microsatellite primers developed for a given species are sometimes useful for another in the same genus and in other genera within the same family, making possible to search for pre-existing suitable primers in the databanks such as GenBank. We examined whether existing primers developed for Polistes could be used for Polistes satan Bequaert. We tested 50 microsatellite primers from three Polistes species and found that six microsatellite loci show polymorphism in size in P. satan. These six loci were highly polymorphic, having four to 15 alleles in P. satan with an expected heterozygosity of 0.525-0.832. These loci can be used to study parameters concerning genetic relatedness such as social interactions in colonies and genetic conflicts of interest among nestmate individuals.

DEVELOPMENT OF MICROSATELLITE PRIMERS FOR JATROPHA CURCAS (EUPHORBIACEAE) AND TRANSFERABILITY TO CONGENERS

Premise of the study: Microsatellite primers were developed for Jatropha curcas (Euphorbiaceae), a tree species with large potential for biofuel production, to investigate its natural genetic diversity and mating system to facilitate the establishment of tree improvement and conservation programs. Methods and Results: Using a protocol for genomic library enrichment, 104 clones containing 195 repeat motifs were identified. Primer pairs were developed for 40 microsatellite loci and validated in 41 accessions of J. curcas from six provenances. Nine loci were polymorphic revealing from two to eight alleles per locus, and six primers were able to amplify alleles in the congeners J. podagrica, J. pohliana, and J. gossypifolia, but not in other Euphorbiaceae species, such as Hevea brasiliensis, Manihot esculenta, or Ricinus communis. Conclusions: The primers developed here revealed polymorphic loci that are suitable for genetic diversity and structure, mating system, and gene flow studies in J. curcas, and some congeners.

COMPARISON OF PRIMERS FOR RAPD-PCR FROM ENVIRONMENTAL ISOLATES OF CRYPTOCOCCUS NEOFORMANS, CRYPTOCOCCUS ALBIDUS AND CRYPTOCOCCUS LAURENTII COMPLEX

Various organisms have been characterized by molecular methods, including fungi of the genus Cryptococcus. The purposes of this study were: to determine the discriminatory potential of the RAPD (Random Amplified Polymorphic DNA) primers, the pattern of similarity of the Cryptococcus species, and discuss their useful application in epidemiological studies. We analyzed 10 isolates of each specie/group: C. albidus, C. laurentii complex, C. neoformans var. grubii, all from environmental source, and two ATCC strains, C. neoformans var. grubii ATCC 90112, and C. neoformans var. neoformans ATCC 28957 by RAPD-PCR using the primers CAV1, CAV2, ZAP19, ZAP20, OPB11 and SEQ6. The primers showed a good discriminatory power, revealing important differences between them and between species; the SEQ6 primer discriminated a larger number of isolates of three species. Isolates of C. laurentii showed greater genetic diversity than other species revealed by all six primers. Isolates of C. neoformans were more homogeneous. Only the primer CAV2 showed no amplification of DNA bands for C. albidus. It was concluded that the use of limited number of carefully selected primers allowed the discrimination of different isolates, and some primers (e. g., CAV2 for C. albidus) may not to be applied to some species.