Littérature scientifique sur le sujet « 5.8S –ITS rDNA »

The unidentified strains AS 2.0706T, preserved in the China General Microbiological Culture Collection Center (CGMCC), Academia Sinica, Beijing, China, and CBS 6904T, preserved in the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands, were shown to represent two novel ascomycetous yeast species of the genus Kazachstania by 18S rDNA, internal transcribed spacer (ITS) region (including 5·8S rDNA) and 26S rDNA D1/D2 domain sequence analysis and electrophoretic karyotype comparison. The names Kazachstania aquatica sp. nov. and Kazachstania solicola sp. nov. are proposed for strains AS 2.0706T and CBS 6904T, respectively. Phylogenetically, the two novel species are closely related to Kazachstania aerobia, Kazachstania servazzii and Kazachstania unispora.

Blastocystisis a common single-celled enteric parasite found in a large variety of hosts. Recent molecular analysis supports the concept that this eukaryotic organism is a stramenopile most closely related toProteromonas lacertae, a parasite of reptiles. In this study, the internal transcribed spacer region, partial small subunit rRNA and large subunit rRNA genes from 7Blastocystisisolates (5 human, 1 pig and 1 sheep), and aProteromonas lacertaeisolate were amplified by PCR, cloned and sequenced.Blastocystiswas found to be a typical eukaryote with both ITS1 and ITS2 regions present. Phylogenetic analysis based on the entire PCR amplicon revealed that theBlastocystisisolates did not segregate according to host or geographic origin. The highest sequence identities with the conservedBlastocystis5·8S rDNA sequence were with the stramenopilesFibrocapsa japonica,Chattonella marina,Cylindrotheca closteriumandHyphochytrium catenoides. The most parsimonious tree based on the 5·8S rDNA sequence fromP. lacertae, 11 other stramenopiles, 2 fungi, 3 algae and 3 alveolates showedBlastocystispositioned within the stramenopiles, withP. lacertaeas its closest relative. This work therefore supports the hypothesis thatBlastocystisis most closely related toP. lacertae, and that it should be regarded as an unusual stramenopile.

Colletotrichum lindemuthianum is the causal agent of anthracnose in common bean. Favorable conditions for this disease might result in up to 100% yield losses. One of the main challenges for common bean producers and breeders still remains the management disease, since this pathogen exhibits a wide genetic variability probably due to its recombination sexual reproduction. The 5·8S gene and the flanking internal transcribed spacer regions (ITS1 and ITS2) of 40 different isolates of C. lindemuthianum collected in Brazil were amplified by PCR, and sequenced in order to determine genetic variability. The results revealed that 46.88% of SNPs were detected in the ITS1 region, while 53.12% of them were located in the ITS2 region. The genetic distance ranged from 0.000 to 0.169 between races. The greatest distance was observed between the races 10 and 73 with a value of 0.169, indicating a wide genetic variability between them. The phylogenetic tree was composed of three groups. Group I had five subgroups. Similar results were also observed through population structure analysis, which revealed the presence of three clusters. These results suggest that sequence analysis of ITS rDNA regions of C. lindemuthianum may be a valuable tool to identify this pathogen through design of specific primers.

The ascaridoid nematode of cats from Kuala Lumpur, Malaysia, previously identified morphologically as Toxocara canis, was characterized using a molecular approach. The nuclear ribosomal DNA (rDNA) region spanning the first internal transcribed spacer (ITS-1), the 5·8S gene and the second internal transcribed spacer (ITS-2) was amplified and sequenced. The sequences for the parasite from Malaysian cats were compared with those for T. canis and T. cati. The sequence data showed that this taxon was genetically more similar to T. cati than to T. canis in the ITS-1, 5·8S and ITS-2. Differences in the ITS-1 and ITS-2 sequences between the taxa (9·4–26·1%) were markedly higher than variation between samples within T. canis and T. cati (0–2·9%). The sequence data demonstrate that the parasite from Malaysian cats is neither T. canis nor T. cati and indicate that it is a distinct species. Based on these data, PCR-linked restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) methods were employed for the unequivocal differentiation of the Toxocara variant from T. canis and T. cati. These methods should provide valuable tools for studying the life-cycle, transmission pattern(s) and zoonotic potential of this parasite.

SummaryThe extent of intra-individual and intra-species heterogeneity in the nuclear rDNA internal transcribed spacer (ITS) was investigated among the ‘Asiatic Vigna’ species (subgenus Ceratotropis). High intra- and inter-individual ITS polymorphism was observed among Vigna radiata accessions, where multiple ITS length variants ranging from ~700 to ~770 bp were detected on PCR amplification. Subsequent analysis revealed that the variants are ‘heteroduplex ITS fragments’ generated during the PCR process. Analysis of ITS from wild and cultivated forms of ten Vigna species from subgenus Ceratotropis revealed substantial intra-species divergence in four species: Vigna umbellata, Vigna trilobata, V. radiata and Vigna minima. However, no other species analysed showed intra-individual ITS heterogeneity as observed in V. radiata. The results demonstrate differential evolution of ITS sequence among wild and cultivated forms of V. radiata. Evidence indicates that intra-species hybridization and a slow ‘molecular drive’ are responsible for this phenomenon. Sequence analysis of 5·8S, ITS1 and ITS2 and secondary-structure analysis of ITS regions indicate that the ITS variants do not belong to pseudogenic rDNA repeat units. Further, reverse transcriptase-PCR (RT-PCR) analysis showed that rDNA repeat units harbouring certain intra-individual ITS variants were transcriptionally inactive, indicating the regulation of these loci by epigenetic gene silencing. The V. radiata ITS variants, when analysed together, did not cause any phylogenetic errors at the species level.

SUMMARYThe taxonomic history of members of the 37-collar-spine group within the genus Echinostoma has been very confused. We obtained DNA sequence data from the nuclear rDNA ITS1, 5·8S and ITS2 of 7 nominal species belonging to this group, Echinostoma trivolvis (Cort, 1914), E. revolution (Frölich, 1802), E. caproni Richard, 1964, E. liei Jeyarasasingam et al. 1972, E. paraensei Lie & Basch, 1967, two African isolates, E. sp.I and E. sp.II, and of one 28-collar-spined echinostome, E. hortense (Asada, 1926). Five of the eight species were clearly distinguishable using ITS data. Sequences from the remaining three taxa, E. caproni, E. sp.II and E. liei were identical to one another and the group containing these taxa was distant from other 37-collar-spine species on a phylogenetic tree. E. trivolvis and E. paraensei form a second, but less distinct group within the 37-collar-spine group. The resolution obtained using DNA sequencing will assist in the current reclassification of the group. It also provides a model for future work on sibling species.

AbstractIn order to identify monophyletic groups within the family Parmeliaceae, eleven taxa (Bryoria capillaris, Cetraria islandica, Evernia pruniastri, Hypogymnia physodes, Parmelia saxatilis, Platismatia glauca, Pleurosticta acetabulum, Usneaflorida, Vulpicida juniperina, V. pinastri, and Xanthoparmelia conspersa) were studied using newly produced nuclear rDNA sequence data from the ITS and 5·8S regions. The resulting evolutionary hypothesis was compared with results from previous phylogenetic analyses based on anatomy, morphology, and chemistry. The outcome of this comparison does not support the earlier proposed phylogenies but is not stable enough for identifying monophyletic groups, with one exception. The results indicate a close relationship between Cetraria and Vulpicida, which is contradictory to previous published analyses. The variation in ascus structures in the Parmeliaceae is discussed and it is questioned whether the earlier distinguished ‘ forms ’ of ascus types represent synapomorphies, if they are based on poorly supported analyses, or if they are exaggerations of relatively slight variation in shape. Further interpretations of the results are discussed and areas of future studies based on DNA-data are suggested.

The Cryptosporidium ITS1, 5·8S and ITS2 rDNA regions from a number of Cryptosporidium isolates from different hosts and geographical areas were cloned and sequenced in order to investigate the extent of sequence heterogeneity between human and cattle-derived isolates from different geographical locations and also between isolates of Cryptosporidium from different hosts such as cats, pigs, mice and a koala. Calf-derived isolates from different continents were virtually identical as were human-derived isolates from the UK and Australia. Genetic differences between Cryptosporidium isolates were extensive and were in fact greater than the level of nucleotide divergence between Toxoplasma gondii and Neospora caninum rDNA sequences. Based on the sequence information derived from this study, PCR–RFLP of the ITS1 region was undertaken in order to directly amplify and genotype Cryptosporidium isolates from different hosts. This PCR–RFLP approach can now be used for molecular epidemiology studies, circumventing the need for costly sequencing and allowing a wider range of genetically different isolates to be examined.

Cryptococcus neoformans (C. neoformans), um fungo que se encontra disseminado em várias partes do mundo, inclusive no Brasil, é o responsável pela criptococose infecção oportunista mais comum em pacientes com a síndrome da imunodeficiência adquirida (SIDA). O caráter sistêmico da criptococose pode levar esses pacientes a óbito. A finalidade de se obter um diagnóstico laboratorial rápido e acurado de C. neoformans, principalmente para o seguimento dos pacientes HIV nos levou a investigar \"seqüências iniciadoras\" (Si) A, B e C das regiões 18SrDNA, 5,8SrDNA e ITS do Cryptococcus spp. Pela Nested PCR com estas seqüências, sugerimos a melhor delas para o desenvolvimento de um diagnóstico molecular em relação aos métodos usuais. Para tal, foram avaliadas amostras de soro e líquor de 39 pacientes, que já haviam recebido tratamento clínico. Todos os casos foram selecionados em grupos, como segue:7 com criptococose (GIII), 14 HIV positivos (GIV), 18 HIV positivos associados com a criptococose (GV) em relação a 10 controles - indivíduos sadios (GI) e amostras de culturas referência (GII). Os resultados obtidos pela Nested PCR com as \"Sis\" A, B e C foram comparados àqueles obtidos pelos métodos de diagnóstico convencionais. As análises desse estudo mostraram que as \"Sis\" A, B e C detectam C. neoformans com especificidade variada, tanto no soro (SiA 91,66%, SiB-100%, SiC 75%), como no líquor (SiA 83,33%, SiB 100% e SiC 75%) mas não apresentaram falso positivo, quando esses resultados foram comparados aos obtidos das culturas heterólogas (GVI). A SiB, em líquor, apresentou sensibilidade, acurácia, valores preditivo positivo e negativo, e especificidade de 100% para a detecção de C. neoformans da mesma forma que no soro, porém neste o valor preditivo negativo foi 89%, acurácia 94% e a sensibilidade 88%. Em amostras de soro e líquor, os testes Tinta da China e Látex, mostraram resultados falso positivos para o GIV e falso negativos nos grupos GIII e GV. As análises comparativas entre as técnicas mostraram que a ordem de eficiência da sensibilidade para detecção de C. neoformans no soro foi SiB>SiA=Látex>SiC e no líquor foi SiB> SiA>tinta da China>Látex=SiC. No soro, a especificidade entre as técnicas foi SiB>SiA=Látex>SiC e no líquor foi SiB=tinta da China>Látex>SiA>SiC. De acordo com nossos dados, concluímos que, independente da doença associada (HIV) ou se o paciente for tratado, a SiB foi a melhor seqüência para a detecção de C. neoformans, tanto em amostras diretamente de soro, como de líquor para todos os grupos estudados. Tendo em vista os fatos, acreditamos que, a aplicação da técnica da Nested PCR com a \"SiB\", em amostras de líquor e soro, é um método viável e acurado para realizar o diagnóstico molecular do C. neoformans em pacientes HIV. O uso de amostras de soro, para o segmento dos pacientes com SIDA, durante o tratamento, pode ser a forma menos invasiva em relação ao líquor para a detecção do C. neoformans, com vantagens sobre os métodos utilizados
Cryptococcus neoformans (C. neoformans), a fungus that is widespread in many parts of the world, including Brazil, is responsible for cryptococcosis the most common opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS). The systemic character of cryptococcosis may be fatal. In order to obtain a rapid and accurate laboratory diagnosis to follow - up of HIV-cryptococcosis patients led us to investigate the sensibility and especificity of three primers (A, B and C) of 18SrDNA, 5,8SrDNA and ITS regions of Cryptococcus spp. Using Nested PCR with those primers we suggest the best among them to be used as a method of molecular diagnosis in relation to the usual techniques. For this purpose, the serum and cerebrospinal fluid (CSF) of 39 patients, who had received medical treatment, were evaluated. All cases were separated in groups, as follows: 7 with cryptococcosis (group III), 14 HIV positive (group IV), 18 HIV positive associated with cryptococcosis (group V) were compared to, 10 healthy subjects (group I) controls, as well as to reference cultures (group II) samples. The results obtained by nested PCR with primers A and B and C were compared to those obtained by conventional diagnostic methods. The analyses of primers A, B and C detected C. neoformans both in serum (SiA 91,6%, SiB-100%, SiC 75%), and in CSF (SiA 83,3%, SiB 100% e SiC 75%). Besides, they were specific for the identification of C. neoformans and showed that there were no false positives when compared with heterologous cultures (group VI) samples. The primer B in CSF showed 100% sensitivity, 100% accuracy and 100% predictive values (positive and negative), and 100% specificity for the detection of C. neoformans, the same that occurs in serum, but in this case, with 88% in sensitivity, 89% predictive value negative and 94% accuracy. In serum and CSF samples the China Ink and the Latex tests showed false positive results for group IV and false negative for III and V groups. The comparative analysis among the techniques (Nested PCR, China Ink, Latex) indicated that the efficiency order of sensitivity for the detection of C. neoformans were PrB> PrA = Latex> PrC in serum and PrB > PrA > China Ink> Latex = PrC in CSF. For the serum and CSF specificities the same techniques were used with the following results: PrB>PrA=Latex>PrC and PrB=China Ink>Latex>PrA>PrC. According to our data we conclude that, whether the patients had been under treatment or not, the Nested PCR by PrB was the best way to detect C. neoformans both in serum and in CSF for all groups. The following up of AIDS patients, throughout the course of therapy was found to be feasible, accurate and less invasive to detect C. neoformans by using serum samples (directly)