Development of a PCR-ELISA assay for diagnosis of Perkinsus marinus and Perkinsus atlanticus infections in bivalve molluscs

Perkinsus atlanticus and P. marinus have been associated with mass mortality of bivalve molluscs. Perkinsus infections are routinely diagnosed by histology or the fluid thioglycollate medium (FTM) assay. In this study, we describe the development of a PCR-enzyme-linked immunosorbent assay (ELISA) for amplification and rapid detection of Perkinsus species. The PCR reactions were selected to either amplify an IGS sequence region shared by currently accepted Perkinsus species or to simultaneously amplify IGS regions specific to either P. atlanticus or P. marinus. The specific hybridisation of DIG-labelled amplified products to species-specific capture probes was detected colorimetrically. This assay is able to specifically detect P. atlanticus and P. marinus, and the intensity of the colorimetric signal is dependent upon the amount of amplified product. The PCR-ELISA assay format is 100-fold more sensitive than visualisation of PCR products on ethidium bromide (EtdBr)-stained agarose gels, and as sensitive as Southern hybridisation. The sensitivity limit of PCR-ELISA was 1 pg of DNA from P. atlanticus. No cross-reactivity of the assay was observed against the host DNA. When applied to the detection of P. atlanticus in clams, 39 samples out of 45 yielded concordant results for FTM assay and PCR-ELISA detection.     

Rapid differentiation of species of Botryosphaeriaceae by PCR fingerprinting

The fingerprinting methods referred to as MSP-PCR (microsatellite-primed polymerase chain reaction) and rep-PCR (repetitive-sequence-based polymerase chain reaction) were used to discriminate between species of Botryosphaeriaceae (Fungi, Ascomycota). Several primers were tested with both methods and each primer enabled clear differentiation of all the species tested. Cluster analysis of banding patterns of the isolates corresponded well with known species delineations based on morphology and phylogenetic analysis. The methods described in this paper provide simple and rapid procedures that can be used for routine differentiation of Botryosphaeriaceae isolates at the species level. The two methods are also useful for studying intraspecific variability.     

Tissue heterogeneity as a mechanism for localized neural stimulation by applied electric fields

We investigate the heterogeneity of electrical conductivity as a new mechanism to stimulate excitable tissues via applied electric fields. In particular, we show that stimulation of axons crossing internal boundaries can occur at boundaries where the electric conductivity of the volume conductor changes abruptly. The effectiveness of this and other stimulation mechanisms was compared by means of models and computer simulations in the context of transcranial magnetic stimulation. While, for a given stimulation intensity, the largest membrane depolarization occurred where an axon terminates or bends sharply in a high electric field region, a slightly smaller membrane depolarization, still sufficient to generate action potentials, also occurred at an internal boundary where the conductivity jumped from 0.143 S m(-1) to 0.333 S m(-1), simulating a white-matter-grey-matter interface. Tissue heterogeneity can also give rise to local electric field gradients that are considerably stronger and more focal than those impressed by the stimulation coil and that can affect the membrane potential, albeit to a lesser extent than the two mechanisms mentioned above. Tissue heterogeneity may play an important role in electric and magnetic 'far-field' stimulation.     

Cavitary disease and quantitative sputum bacillary load in cases of pulmonary tuberculosis

We examined sputum bacterial loads in adults with newly diagnosed tuberculosis using quantitative culture and time-until-positive (DTP) culture in BACTEC 460. Patients with cavitary disease had higher CFU levels than those without cavities and shorter DTPs. Within radiographic strata of moderately and far advanced tuberculosis, higher CFU counts were associated with cavitary disease.     

Anticipating rotavirus vaccines in Brazil: detection and molecular characterization of emerging rotavirus serotypes G8 and G9 among children with diarrhoea in Recife, Brazil

In 2006, Brazil will initiate universal immunization of its 4-million infants with a live attenuated serotype G1P[8] human rotavirus vaccine. In anticipation of the national immunization program, this study was undertaken to characterize rotavirus strains circulating among children in Recife, one of the largest cities in the northeast region of Brazil. Group A rotaviruses were detected in 102 (35%) of 290 faecal specimens collected from children under 5 years of age who presented with acute diarrhoea during a 1-year period between May 2004 and April 2005. In addition to the globally common G1P[8] serotype that accounted for 49% of strains, emerging rotavirus serotypes G8P[6] and G9P[8] represented 2% and 29% of strains, respectively. Following cell culture adaptation, RNA-RNA hybridization demonstrated that two Brazilian G8P[6] rotavirus strains shared a high level of genomic RNA homology with Malawian G8P[6] strains, and a Brazilian G9P[8] strain was related most closely to a G9P[8] strain from India. The results suggest that certain rotavirus strains have a much wider global circulation than generally appreciated. Continued global spread of such strains might challenge the efficacy of current rotavirus vaccines.     

Hantavirus infection in Brazil: development and evaluation of an enzyme immunoassay and immunoblotting based on N recombinant protein

The symptoms of hantavirus pulmonary syndrome may resemble those of other febrile illnesses. The development of an accurate diagnostic test should therefore improve clinical prognosis and be useful in epidemiologic studies. We evaluated the use of a recombinant antigen (rNDelta(85)) based on the S-segment sequences of a Brazilian hantavirus for detecting immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against hantavirus in an indirect enzyme immunoassay (EIA). We assayed 613 serum samples (570 from humans and 43 from rodents). IgM EIA had a sensitivity of 94.1% and a specificity of 99.1%. IgG EIA had a sensitivity of 95.2% and a specificity of 98.4%. This evaluation confirms that rNDelta(85) IgM and IgG EIA tests are potentially useful rapid, sensitive, and cost-effective tools for detecting antibodies against hantaviruses indigenous to Brazil and other South American countries, in patients with acute or convalescent hantavirus infection, and in rodent reservoirs.     

Low-frequency noise effects on the parotid gland of the Wistar rat

BACKGROUND: Long-term low-frequency noise (LFN) (相似文献   

Clinical effectiveness of two microabrasion materials for the removal of enamel fluorosis stains

This study evaluated the effectiveness of two microabrasion products for the removal of enamel fluorosis stains. Using a split-mouth study design, two operators used PREMA (PM) and Opalustre (OP) to remove fluorosis-like stains from 36 subjects (10-12 years old). Both products were rubbed onto the surface of the affected teeth for 30 seconds. This procedure was repeated five times during each clinical appointment. A maximum of three clinical appointments were scheduled. The subjects and/or their parents were questioned about their satisfaction with the treatment. Two blinded evaluators appraised both sides of the mouth using a visual scale system. The data were analyzed by Friedman repeated measures ANOVA and Wilcoxon test. The majority of the subjects (approximately 97%) reported satisfaction at the end of the treatment (p = 0.0001). A significant improvement in appearance was detected after the second clinical appointment when using PREMA and Opalustre (p < 0.002). After the first clinical appointment, OP showed a statistically higher mean rating for improvement in appearance (3.4 +/- 0.7) than PM (2.4 +/- 0.5) (p = 0.002).