To overcome the limitations of the presumed universalprimers, new primer pairs have been developed targeting specific large assemblages, such as birds , lepidop- terans , or fish . Still, the limited amplification suc- cess in some groups led to the development of alternative approaches, namely the design of degenerate primer pairs  or even primer cocktails . The later approach has been very successful in COI-5P amplification in fish (e.g. ), although one of the primer cocktails was originally designed and tested in mammals, and the same occurred for the alternative primer pairs designed for birds  or Lepidoptera . Despite the success of these group- specific PCR primers, to date no alternatives to Folmer primers have been proposed that are effective in a broad range of marine animals and particularly for marine inver- tebrates. Within the latter group, most PCR primers devel- oped were phylum or class specific (see ), like primers designed for Echinoderms , Crustacea (, D. Steinke unpublished in ), Gastropoda  and Annelids . Here we propose a new pair of enhanced primers specific- ally designed to amplify the COI-5P barcode region from a broad taxonomic range of marine organisms. We com- pared its amplification potential with other broadly used primer pairs, and tested amplification success in 76 species from both vertebrates and invertebrates and a total of 8 animal phyla. The success of amplification in a broad- range of taxa indicates that these primers can be particularly valuable for building up reference DNA barcode libraries of complex marine communities.
Universal oligonucleotides evaluation in the semi-nested RT-PCR of the IBV S1 gene Two degenerate primers (SYU+, SYU-) complementary to S1 regions and an anti-sense universal primer complementary to the beginning of S2 gene were used as universal primers in the semi- nested RT-PCR. This procedure allowed for a general amplification of the major part of S1 gene (1,737 bp) in the first PCR, followed by the amplification of a smaller fragment of S1 (451bp) in the semi-nested PCR. Figure 2 shows that a fragment with the expected size (451 bp) was amplified when all four IBV strains were tested (H120, M41, Arkansas and A034).
geographic distribution and genetic relationships among the studied penaeid species and the implications for conservationist approaches. Specific oligonucleotides were designed to amplify four mtDNA regions (COI, 16S, Cytb and DLoop). The results showed efficient patterns for 16 native species of Brazilian and Mozambican coasts. Numts presence and its implication for penaeid genetics were investigated using specific and universalprimers for COI gene. Pseudogenes were detected for some penaeid species. Cytb pseudogene for the Penaeidae family was reported by the first time. DNA barcoding approach was tested in several species from Brazilian coast. The barcoding analysis evidenced a high range of intraspecific distance, suggesting the necessity of taxonomic review into Penaeidae. The existence of species complex was investigated for both X. kroyeri and F. subtilis. Phylogeographical signs and population structure were no observed for F. subtilis along the Brazilian coast. F. paulensis populations, collected in Lagoa dos Patos (RS) and Cananéia (SP), showed genetic structuring by analyzing of COI gene. That data can be reflecting the existence of differentiated-genetically adult stocks or ancient structure. Two recent population expansions, one of them before and the other one after the last period of the maximum glacial, were observed for F. paulensis. mtDNA and RAD-seq data were using to study the species X. kroyeri populations. The
The first dengue fever epidemic in the State of Rondônia (western region of Brazil) was recorded in 1997, without laboratory confirmation. Following this, there was an epidemic in Manaus, in the neighboring State of Amazon, in 1998, in which DENV-1 and DENV-2 viruses were isolated from patients. In the present paper, the serotype characterization of the dengue virus isolated from patients with clinically suspected dengue in Porto Velho, Rondônia, between 2001 and 2003 is described. One hundred and fifty blood samples were collected between the first and fifth days of symptoms. Seventy samples of virus isolates were subjected to dengue identification by means of RT-PCR using universalprimers for the NS1 gene of DENV, which amplifies a 419 bp fragment. The amplicons obtained were subjected to enzymatic digestion to characterize the viral serotypes. All the samples analyzed were DENV-1. A nucleotide sequence randomly selected from one amplicon, which was also DENV-1, presented 98% similarity to sequences from Southeast Asia that were obtained from GenBank.
sensitivity. This amplification step can be easily included if the pathogens targeted have a conserved gene for which generic primers are designed, as for the 16S rRNA gene for prokaryotes or mitochondrial genes for eukaryotes. However this is not feasible for viruses from unrelated taxonomic groups due to the inexistence of conserved genes. This issue was overcome by uncoupling the specific target recognition from amplification. One strategy has been to use Padlock probes - also known as circularizable probes - (Thomas et al. 1999) for target recognition. These probes have two pathogen specific regions at the 5 and 3’ ends that come together upon hybridization to the target and are ligated by a DNA ligase. The resulting closed circular molecule becomes permanently locked around the target strand, like a padlock, enabling very stringent washings. The probe has also one or two regions between the pathogen specific ones that will function as universalprimers in a subsequent amplification by Rolling Circle Amplification or PCR and a zip-code sequence for hybridization with a subset of the probes in the microarray (Fig. 4). This strategy has been used for fungi and nematodes (Szemes et al. 2005; van Doorn et al. 2007). In another strategy, Engel et al. (2010) used a random primed PCR amplification before hybridization in the development of a microarray designed to detect 44 viruses of the grapevine. To avoid the competing simultaneous amplification of host rRNA, those authors used an enriched ds-RNA preparation instead of total RNA. A comparison of available alternatives for amplification prior to hybridization can be found elsewhere (Vora et al. 2004; Vora et al. 2008). A variety of formats for the arrays and equipment for constructing the array and register the signal exist including electronic solutions in which hybridization is monitored through changes in impedance - for a review see Ramanavicius and Ramanaviciene (2007). However the diversity of existing formats does not help to decrease the price of the equiment and the transfer of microarrays for routine diagnosis is not at close sight.
subjected to shear bond strength test (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 universalprimers on control and air-abraded zirconia surfaces. Data (MPa) were analyzed using one-way ANOVA and Tukey`s, and Student’s T-tests (alpha=0.05). Results: When universalprimers were used alone, SbU presented significantly higher mean SBS (19.49±5.84) that those of other primers (0-9.93±6.61) (p=0.001). When air-abraded, the groups AP-A (14.06±6.05) ab , MP-A
Polymerase chain reaction (PCR) was performed using UTSP as described by Ribeiro et al. (2013). This ge- notyping system was originally proposed by Oetting et al. (1995), which used a fluorescently labeled primer to an- neal with a specific primer used to amplify the target frag- ments in PCR, labelling indirectly the amplified fragment. The UTSP system was composed of three primers: the sense microsatellite primer with 17 extra base pairs (bp) tail in the 5’ end, these additional sequences are identical to the universalprimers T3 (ATTAACCCTCACTAAAG), T7 (AATACGACTCACTATAG) or M13 (GTAAAACGACG- GCCAGT); the respective antisense microsatellite primer, and the universal primer with the same sequences of the sense primer tails, which can be T3, T7 or M13 labeled with each of the fluorochromes HEX, NED or 6-FAM, re- spectively. The amplification reaction (20 µL) contained DNA (30 ng), 1X PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.08 % Nonidet P40), 1.5 mM MgCl 2 , 0.25 mM dNTP, 0.025 µM of sense primer, 0.125 µM of antisense primer, 0.125 µM of labeled universal primer and 1.0 U of Taq DNA polymerase. Conventional amplification reac- tions were performed using the same conditions applied to the UTSP, except for the primers, which were at 0.125 µM for the regular sense and antisense SSR primers. The PCR for each microsatellite locus was performed sepa- rately and mixed before loading in the sequencer.
The nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) from leaves of Drosera (Droseraceae) were amplified using “universal” primers. The analysis of the products demonstrated most samples were a molecular mixture as a result of unsuccessful and non-specific amplifications. Among the obtained sequences, two were from Basidiomycota fungi. Homologous sequences of Basidiomycota were obtained from GenBank database and added to a data set with sequences from Drosera leaves. Parsimony analysis demonstrated that one sequence was amplified from an Ustilaginomycetes fungus, and another from a Heterobasidiomycetes. Possibly these fungi were associated to leaves of Drosera, and not because of samples contamination. In order to provide optimization and a better specificity of PCR (polymerase chain reaction), a very successful method was demonstrated using dimethyl sulfoxide (DMSO) and bovine serum albumin (BSA) in reactions.
For the UTSP method, a multiplex system was used, as described by Oetting et al. (1995), which included three kinds of primers: 1, a sense microsatellite primer, which carries, besides its specific sequence, a 17 base pair (bp) tail at its 5’ end identical to sequences of universalprimers commonly used in molecular biology, such as: M13 (GTAAAACGACGGCCAGT), T3 (ATTAAC CCTCACTAAAG) or 7 (AATACGACTCACTATAG); 2, a regular antisense microsatellite primer; and 3, a fluorescently labeled primer, with the same sequence as the sense primer tail (Figure 1). The tails were tagged with either 6‑FAM (M13 tail), HEX (T3 tail) or NED (T7 tail) (Life Technologies do Brasil Ltda., São Paulo, SP, Brazil).
Eight out of 21 Fabaceae-specific primer pairs (38.09%) appear to be suitable for members of Cucurbitaceae, Euphorbiaceae, Rosaceae and A. thaliana. The performance of some of these primers may be improved by the replacement of up to two nucleotides in their primary nucleotide sequences to increase their complementarity with template DNA. This would imply that the development of more universalprimers was possible via the introduction of degenerate nucleotides. However, this was avoided because degenerate primers are likely to have additional (multiple) binding sites especially in species with large mitogenomes, as observed for several degenerate primers published by DUMINIL et al. (2002). On the other hand, the assessment of transferability of Fabaceae-specific primers into more or less related species revealed an interesting trend. That is, although higher transferability rates are expected into more closely related species, the size of the mtDNA genome apparently plays an important role in this aspect as well. The highest transferability of primers was observed in Citrullus lanatus (Cucurbitaceae) having a rather small mtDNA genome, while transferability into a less related A. thaliana, characterized also by a small mtDNA genome, was higher than into other members of Cucurbitaceae (Cucurbita pepo, Cucumis sativus mitochondrion chromosome 1, and Cucumis melo subsp. melo) having mtDNA genomes of 1 Mbp or larger. This would suggest that the size of mtDNA genomes may also be used as an indicator of the potential transferability of Fabaceae- specific primers into more or less related taxa.
There is a substantive body of international evidence about the positive impact of APN roles for improving patient health outcomes, quality of care and health system eficiency. The implementation of these roles can address country needs to improve universal health coverage and universal access to health in Latin America and the Caribbean. Several middle and high income Latin American countries with access to graduate nursing education are posed to introduce these roles. Other important elements in place to support APN role introduction in these countries include the alignment of APN outcomes with health care policies for primary health care reform and a developing coalition of nursing leaders across health care, academic, and health policy sectors both within and external to Latin American countries. Further expansion to engage other intersectoral leaders will be required to move the APN agenda forward.
The slope of a standard curve is mathematically correlated to PCR efficiency according to the equation E = 10 21/slope 21, where E is the PCR efficiency . A 100% efficiency corresponds to a slope value of 23.32. Slopes of the real-time DOP-PCR experiments ranged from 23.9 to 24.1 depending on the species of template DNA. These slope values correspond to amplification efficiencies of 70 to 80% in DOP-PCR which are lower than those in ordinary real-time PCR. This means that about 20 to 30% of template DNA molecules failed to serve as successful templates to produce new DNA molecules in each PCR cycle. This might have resulted from decreased priming efficiency of perfect match primers to templates by competition with partially complementary primers. Since random sequences are included in the DOP primer, there will exist excessive amounts of partially complemen- tary primers, which could compete and interfere with the perfect match primers for priming. Another possibility is self-annealing of template DNA molecules. In contrast to ordinary PCR, all amplicons have the same primer sequences at the both ends except for random sequences. Therefore, the possibility of self-annealing among denatured template molecules would be greatly elevated in DOP-PCR, which could lead to decreased amplification efficiency. However, in spite of the lower amplification efficiencies of DOP- PCR, standard curves of all DNA species exhibited inter- experimental variations less than 0.5 as C t values that correspond
O presente artigo resultou de um trabalho realizado sobre uma biblioteca virtual e sua importância para a preservação da memória. A Biblioteca Digital Universal (BDU) é uma biblioteca em âmbito mundial originária da Índia que agrega importantes obras (acervos) literárias, artísticas e científicas. Os elementos abordados para este trabalho foram: histórico, missão, objetivos, estrutura da organização e formação do acervo entre outros tópicos considerados relevantes.
Foram analisadas 28 amostras de medula óssea, de 27 pacientes cujos dados clínicos são apresentados nas tabelas 1 e 2. Destas 28 amostras, 11 eram de homens (39%) e 17 eram de mulheres (61%). Todos os pacientes foram estudados citogeneticamente para evidenciar a presença do cromossomo Ph (Tabela 1). As am o st r as d e m RN A f o r am t r an scr i t as reversamente e amplificadas utilizando primers esp ecífi cos p ara detecção dos transcri tos quiméricos b2a2 ou b3a2.
Nunca, como nos anos mais recentes, se discorreu tanto sobre o conceito de biblioteca e se cruzaram tantos argumentos sobre a sua natureza e formas de realização, entre a visão eufórica da biblioteca virtual universal e a ideia de biblioteca ligada a realidades locais e institucionais (que muitos consideram anacrónica). Para isso, não terá deixado de contribuir a disseminação das tecnologias da informação, a crescente transição de dados analógicos para dados digitais e, portanto, o aumento exponencial do nosso espaço digital. Há cada vez mais informação disponível sob forma electrónica, e os seus recursos transformam-se em recursos de informação em rede, no sentido em que, de um ou de outro modo, vêm a encontrar-se disponíveis na Internet. Essa dimensão tecnológica caracteriza-se agora por uma expansão sem precedentes, o que, não deixando de influenciar alguns dos atributos da biblioteca, pode permitir que dela se transmita uma visão superficial, utópica e descarnada, de que a expressão “Click – whatever you want is there” é bem ilustrativa. A vulgarização e a mediatização da ideia de biblioteca universal em linha, a que a expansão da Web conferiu renovada ênfase, sendo naturalmente assumida pelos tecnófilos, é, por vezes, estranhamente aceite por pensadores tão inesperados como George Steiner, que chega a considerar a Internet como a “library of libraries” e a prever a “realização do sonho leibniziano de uma Bibliotheca Universalis” graças à disponibilização “da totalidade do registo da memória e do conhecimento humanos no terminal de um computador”.