Identification of species related to Anopheles (Nyssorhynchus) albitarsis by random amplified polymorphic DNA-Polymerase chain reaction (Diptera: Culicidae)

Species-specific Random Amplified Polymorphic DNA-Polymerase chain Reaction (RAPD-PCR) markers were used to identify four species related to Anopheles (Nyssorhynchus) albitarsis Lynch-Arribàlzaga from 12 sites in Brazil and 4 in Venezuela. In a previous study (Wilkerson et al. 1995), which included sites in Paraguay and Argentina, these four species were designated "A", "B", "C" and "D". It was hypothesized that species A is An. (Nys.) albitarsis, species B is undescribed, species C is An. (Nys) marajoara Galvão and Damasceno and species D is An. (Nys.) deaneorum Rosa-Freitas. Species D, previously characterized by RAPD-PCR from a small sample from northern Argentina and southern Brazil, is reported here from the type locality of An. (Nys.) deaneorum, Guajará-Mirim, state of Rondônia, Brazil. Species C and D were found by RAPD-PCR to be sympatric at Costa Marques, state of Rondônia, Brazil. Species A and C have yet to be encountered at the same locality. The RAPD markers for species C were found to be conserved over 4,620 km; from Iguape, state of São Paulo, Brazil to rio Socuavo, state of Zulia, Venezuela. RAPD-PCR was determined to be an effective means for the identification of unknown species within this species complex.

Widespread occurrence of non-canonical transcription termination by human RNA polymerase III.

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3'-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T(≥4) stretch within 50 bp of 3'-flanking region. In vitro analysis of tDNAs with a distanced T(≥4) revealed the existence of non-canonical terminators resembling degenerate T(≥5) elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3' trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3'-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3'-trailer sequences with the potential to contribute novel functional ncRNAs.

Quantitative real-time polymerase chain reaction for detection of adenovirus after T cell-replete hematopoietic cell transplantation: viral load as a marker for invasive disease.

BACKGROUND: The value of adenovirus plasma DNA detection as an indicator for adenovirus disease is unknown in the context of T cell-replete hematopoietic cell transplantation, of which adenovirus disease is an uncommon but serious complication. METHODS: Three groups of 62 T cell-replete hematopoietic cell transplant recipients were selected and tested for adenovirus in plasma by polymerase chain reaction. RESULTS: Adenovirus was detected in 21 (87.5%) of 24 patients with proven adenovirus disease (group 1), in 4 (21%) of 19 patients who shed adenovirus (group 2), and in 1 (10.5%) of 19 uninfected control patients. The maximum viral load was significantly higher in group 1 (median maximum viral load, 6.3x10(6) copies/mL; range, 0 to 1.0x10(9) copies/mL) than in group 2 (median maximum viral load, 0 copies/mL; range, 0 to 1.7x10(8) copies/mL; P<.001) and in group 3 (median maximum viral load, 0 copies/mL; range 0-40 copies/mL; P<.001). All patients in group 2 who developed adenoviremia had symptoms compatible with adenovirus disease (i.e., possible disease). A minimal plasma viral load of 10(3) copies/mL was detected in all patients with proven or possible disease. Adenoviremia was detectable at a median of 19.5 days (range, 8-48 days) and 24 days (range, 9-41 days) before death for patients with proven and possible adenovirus disease, respectively. CONCLUSION: Sustained or high-level adenoviremia appears to be a specific and sensitive indicator of adenovirus disease after T cell-replete hematopoietic cell transplantation. In the context of low prevalence of adenovirus disease, the use of polymerase chain reaction of plasma specimens to detect virus might be a valuable tool to identify and treat patients at risk for viral invasive disease.

A low stringency polymerase chain reaction approach to the identification of Biomphalaria glabrata and B. tenagophila, intermediate snail hosts of Schistosoma mansoni in Brazil

The low stringency-polymerase chain reaction (LS-PCR) with a pair of specific primers for the amplification of the 18S rRNA gene was evaluated as a means of differentiating between the two Schistosoma mansoni intermediate host species in Brazil: Biomphalaria glabrata and B. tenagophila. Individual snails obtained from different states of Brazil were used and the amplification patterns obtained showed a high degree of genetic variability in these species. Nevertheless, 4 and 3 clearly defined specific diagnostic bands was observed in individuals from B. glabrata and B. tenagophila respectively. The detection of snail specific diagnostic bands suggests the possibility of reliable species differentiation at the DNA level using LS-PCR.

Specific Identification of Biomphalaria tenagophila and Biomphalaria occidentalis Populations by the Low Stringency Polymerase Chain Reaction

Although Biomphalaria occidentalis and B. tenagophila are indistinguishable on the basis of shell morphology and the majority of their genital organs, only the latter is susceptible to infection with Schistosoma mansoni. Thus, the identification of these species is fundamental to epidemiological studies of schistosomiasis. Here we describe a simple and rapid method for differentiating B. tenagophila from B. occidentalis based on low stringency polymerase chain reaction and using a pair of primers specific for the amplification of the 18S rRNA gene. Analysis of the low stringency product profiles of populations of these snails from different geographical regions confirmed this approach as being applicable to the identification of B. tenagophila and B. occidentalis in cases where classical morphology is inconclusive

Analysis of Respiratory Syncytial Virus in Clinical Samples by Reverse Transcriptase-Polymerase Chain Reaction Restriction Mapping

The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed

A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification

We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR) for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique

Characteristic bimodal profiles of RNA polymerase II at thousands of active mammalian promoters.

BACKGROUND: In mammals, ChIP-seq studies of RNA polymerase II (PolII) occupancy have been performed to reveal how recruitment, initiation and pausing of PolII may control transcription rates, but the focus is rarely on obtaining finely resolved profiles that can portray the progression of PolII through sequential promoter states. RESULTS: Here, we analyze PolII binding profiles from high-coverage ChIP-seq on promoters of actively transcribed genes in mouse and humans. We show that the enrichment of PolII near transcription start sites exhibits a stereotypical bimodal structure, with one peak near active transcription start sites and a second peak 110 base pairs downstream from the first. Using an empirical model that reliably quantifies the spatial PolII signal, gene by gene, we show that the first PolII peak allows for refined positioning of transcription start sites, which is corroborated by mRNA sequencing. This bimodal signature is found both in mouse and humans. Analysis of the pausing-related factors NELF and DSIF suggests that the downstream peak reflects widespread pausing at the +1 nucleosome barrier. Several features of the bimodal pattern are correlated with sequence features such as CpG content and TATA boxes, as well as the histone mark H3K4me3. CONCLUSIONS: We thus show how high coverage DNA sequencing experiments can reveal as-yet unnoticed bimodal spatial features of PolII accumulation that are frequent at individual mammalian genes and reminiscent of transcription initiation and pausing. The initiation-pausing hypothesis is corroborated by evidence from run-on sequencing and immunoprecipitation in other cell types and species.

Diagnosis of Dengue by Using Reverse Transcriptase-Polymerase Chain Reaction

A rapid identification of dengue viruses from clinical samples by using a nested reverse transcriptase-polymerase chain reaction (RT-PCR) procedure was carried out for diagnostic and epidemiological purposes. RT-PCR identified DEN-1 and DEN-2 viruses in 41% (41/100) of previously confirmed cases and provided an accurate confirmation of DHF in four fatal cases. RT-PCR was also useful for detecting and typing dengue viruses in suspected cases, allowing a rapid identification of new serotypes in endemic areas

Molecular identification of similar species of the genus Biomphalaria (Mollusca: Planorbidae) determined by a polymerase chain reaction-restriction fragment length polymorphism

The freshwater snails Biomphalaria straminea, B. intermedia, B. kuhniana and B. peregrina, are morphologically similar; based on this similarity the first three species were therefore grouped in the complex B. straminea. The morphological identification of these species is based on characters such as vaginal wrinkling, relation between prepuce: penial sheath:deferens vas and number of muscle layers in the penis wall. In this study the polymerase chain reaction restriction fragment length polymorphism technique was used for molecular identification of these molluscs. This technique is based on the amplification of the internal transcribed spacer regions ITS1 e ITS2 of the ribosomal RNA gene and subsequent digestion of these fragments by restriction enzymes. Six enzymes were tested: Dde I, Mnl I, Hae III, Rsa I, Hpa II e Alu I. The restriction patterns obtained with DdeI presented the best profile for separation of the four species of Biomphalaria. The profiles obtained with all the enzymes were used to estimate the genetic distances among the species through analysis of common banding patterns.