983 resultados para PCR detection
Resumo:
To establish a noncontagious control for the Ray thioglycollate test for the detection of Perkinsus in mollusks we evaluated nonviable stages of P. olseni for enlargement of hypnospores and blue/black iodine stain. Trophozoites made nonviable with formalin, irradiation or colchicine failed to swell in thioglycollate. They remained small and did not differentially stain in iodine. Trophozoites that had already developed into hypnospores in thioglycollate were rendered inactive by freezing, ethanol or formalin immersion. They retained their iodinophilic properties and thus could provide a partial control for the Ray Test.
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Cervical auscultation is in the process of gaining clinical credibility. In order for it to be accepted by the clinical community, the procedure and equipment used must first be standardized. Takahashi et al. [Dysphagia 9:54-62, 1994] attempted to provide benchmark methodology for administering cervical auscultation. They provided information about the acoustic detector unit best suited to picking up swallowing sounds and the best cervical site to place it. The current investigation provides contrasting results to Takahashi et al. with respect to the best type of acoustic detector unit to use for detecting swallowing sounds. Our study advocates an electret microphone as opposed to an accelerometer for recording swallowing sounds. However, we agree on the optimal placement site. We conclude that cervical auscultation is within reach of the average dysphagia clinic.
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The flock-level sensitivity of pooled faecal culture and serological testing using AGID for the detection of ovine Johne's disease-infected flocks were estimated using non-gold-standard methods. The two tests were compared in an extensive field trial in 296 flocks in New South Wales during 1998. In each flock, a sample of sheep was selected and tested for ovine Johne's disease using both the AGID and pooled faecal culture. The flock-specificity of pooled faecal culture also was estimated from results of surveillance and market-assurance testing in New South Wales. The overall flock-sensitivity of pooled faecal culture was 92% (95% CI: 82.4 and 97.4%) compared to 61% (50.5 and 70.9%) for serology (assuming that both tests were 100% specific). In low-prevalence flocks (estimated prevalence
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Current methods used to genotype point mutations in Plasmodium falciparum genes involved in resistance to antifolate drugs include restriction digestion of PCR products, allele-specific amplification or sequencing. Here we demonstrate that known point mutations in dihydrofolate reductase and dihydropteroate synthase can be scored quickly and accurately by single-nucleotide primer extension and detection of florescent products on a capillary sequencer. We use this method to genotype parasites in natural infections from the Thai-Myanmar border. This approach could greatly simplify large-scale screening of resistance mutations of the type required for evaluating and updating antimalarial drug treatment policies. The method can be easily adapted to other P. falciparum genes and will greatly simplify scoring of point mutations in this and other parasitic organisms. © 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.
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Objectives: To study the influence of different diagnostic criteria on the prevalence of diabetes mellitus and characteristics of those diagnosed. Design and setting: Retrospective analysis of data from the general-practice-based Australian Diabetes Screening Study (January 1994 to June 1995). Participants: 5911 people with no previous diagnosis of diabetes, two or more symptoms or risk factors for diabetes, a random venous plasma glucose (PG) level > 5.5 mmol/L and a subsequent oral glucose tolerance test (OGTT) result. Main outcome measure: Prevalence of undiagnosed diabetes based on each of three sets of criteria: 1997 criteria of the American Diabetes Association (ADA), 1996 two-step screening strategy of the Australian Diabetes Society (ADS) (modified according to ADA recommendations about lowered diagnostic fasting PG level), and 1999 definition of the World Health Organization (WHO). Results: Prevalence estimates for undiagnosed diabetes using the American (ADA), Australian (ADS) and WHO criteria (95% CI) were 9.4% (8.7%-10.1%), 16.0% (15.3%-16.7%) and 18.1% (17.1%-19.1%), respectively. People diagnosed with diabetes by fasting PG level (common to all sets of criteria) were more likely to be male and younger than those diagnosed only by 2 h glucose challenge PG level (Australian and WHO criteria only). The Australian (ADS) stepwise screening strategy detected 88% of those who met the WHO criteria for diabetes, including about three-quarters of those with isolated post-challenge hyperglycaemia. Conclusion: The WHO criteria (which include an OGTT result) are preferable to the American (ADA) criteria (which rely totally on fasting PG level), as the latter underestimated the prevalence of undiagnosed diabetes by almost a half. The Australian (ADS) strategy identified most of those diagnosed with diabetes by WHO criteria.
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The N-methyl-D-aspartate (NMDA)-selective subtype of ionotropic glutamate receptor is of importance in neuronal differentiation and synapse consolidation, activity-dependent forms of synaptic plasticity, and excitatory amino acid-mediated neuronal toxicity [Neurosci. Res. Program, Bull. 19 (1981) 1; Lab. Invest. 68 (1993) 372]. NMDA receptors exist in vivo as tetrameric or pentameric complexes comprising proteins from two families of homologous subunits, designated NR1 and NR2(A-D) [Biochem. Biophys. Res. Commun. 185 (1992) 826]. The gene coding for the human NR1 subunit (hNR1) is composed of 21 exons, three of which (4, 20 and 21) can be differentially spliced to generate a total of eight distinct subunit variants. We detail here a competitive RT-PCR (cRT-PCR) protocol to quantify endogenous levels of hNR1 splice variants in autopsied human brain. Quantitation of each hNR1 splice variant is performed using standard curve methodology in which a known amount of synthetic ribonucleic acid competitor (internal standard) is co-amplified against total RNA. This method can be used for the quantitation of hNR1 mRNA levels in response to acute or chronic disease states, in particular in the glutamatergic-associated neuronal loss observed in Alzheimer's disease [J. Neurochem. 78 (2001) 175]. Furthermore, alterations in hNR1 mRNA expression may be reflected at the translational level, resulting in functional changes in the NMDA receptor. (C) 2003 Elsevier Science B.V. All rights reserved.
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We have developed a competitive RT-PCR assay, adapted from Lewohl et al. [Brain Res. Brain Res. Protoc. 1 (1997) 347]. for the quantitation of GABA, receptor beta isoforms in human brain using an internal standard that shares high sequence homology to the targets. The internal standard is identical to the beta(1) sequence except for a 61 bp deletion and the incorporation of a Hind III restriction enzyme site. Unlike traditional competitive RT-PCR, which requires a range of internal standard concentrations to be titrated against a constant amount of unknown, this method relies on a standard curve for quantitation of each sample and thus permits increased sample throughput. This method is suitable for the quantitation of beta(1), beta(2) and beta(3) isoforms of the GABA(A) receptor in human alcoholic and control brain. (C) 2003 Elsevier Science B.V. All rights reserved.
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Despite extensive efforts to confirm a direct association between Chlamydia pneumoniae and atherosclerosis, different laboratories continue to report a large variability in detection rates. In this study, we analyzed multiple sections from atherosclerotic carotid arteries from 10 endartectomy patients to determine the location of C. pneumoniae DNA and the number of sections of the plaque required for analysis to obtain a 95% confidence of detecting the bacterium. A sensitive nested PCR assay detected C. pneumoniae DNA in all patients at one or more locations within the plaque. On average, 42% (ranging from 5 to 91%) of the sections from any single patient had C. pneumoniae DNA present. A patchy distribution of C. pneumoniae in the atherosclerotic lesions was observed, with no area of the carotid having significantly more C. pneumoniae DNA present. If a single random 30-mum-thick section was tested, there was only a 35.6 to 41.6% (95% confidence interval) chance of detecting C. pneumoniae DNA in a patient with carotid artery disease. A minimum of 15 sections would therefore be required to obtain a 95% chance of detecting all true positives. The low concentration and patchy distribution of C. pneumoniae DNA in atherosclerotic plaque appear to be among the reasons for inconsistency between laboratories in the results reported.
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Detection of a circumferential crack in a hollow section beam is investigated using coupled response measurements. The crack section is represented by a local flexibility matrix connecting two undamaged beam segments. This matrix defines the relationship between the displacements and forces across the crack section and is derived by applying fundamental fracture mechanics theory. The suitability of the mode coupling methodology is first demonstrated analytically. Laboratory test results are then presented for circular hollow section beams with artificially generated cracks of varying severity. It is shown that this method has the potential as a damage detection tool for mechanical structures. (C) 2002 Elsevier Science Ltd. All rights reserved.
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We report the development of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) for the rapid detection of serum antibodies to West Nile virus (WNV) in taxonomically diverse North American avian species. A panel of flavivirus-specific monoclonal antibodies (MAbs) was tested in blocking assays with serum samples from WNV-infected chickens and crows. Selected MAbs were further tested against serum samples from birds that represented 16 species and 10 families. Serum samples were collected from birds infected with WW or Saint Louis encephalitis virus (SLEV) and from noninfected control birds. Serum samples from SLEV-infected birds were included in these experiments because WNV and SLEV are closely related antigenically, are maintained in similar transmission cycles, and have overlapping geographic distributions. The ELISA that utilized MAb 3.11126 potentially discriminated between WW and SLEV infections, as all serum samples from WNV-infected birds and none from SLEV-infected birds were positive in this assay. Assays with MAbs 2132 and 6B6C-1 readily detected serum antibodies in all birds infected with WNV and SLEV, respectively, and in most birds infected with the other virus. Two other MAbs partially discriminated between infections with these two viruses. Serum samples from most WNV-infected birds but no SLEV-infected birds were positive with MAb 3.676, while almost all serum samples from SLEV-infected birds but few from WNV-infected birds were positive with MAb 6B5A-5. The blocking assays reported here provide a rapid, reliable, and inexpensive diagnostic and surveillance technique to monitor WNV activity in multiple avian species.
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Objective To develop and validate specific, sensitive and rapid diagnostic tests using RT-PCR for the detection of Ross River virus (RRV), Kunjin virus (KV) and Murray Valley encephalitis virus (MVEV) infections in horses. Methods Primer sets based on nucleotide sequence encoding the envelope glycoprotein E2 of RRV and on the nonstructural protein 5 (NS5) of KV and MVEV were designed and used in single round PCRs to test for the respective viruses in infected cell cultures and, in the case of RRV, in samples of horse blood and synovial fluid. Results The primer pairs designed for each of the three viruses amplified a product of expected size from prototype viruses that were grown in cell culture. The identity of each of the products was confirmed by nucleotide sequencing indicating that in the context used the RT-PCRs were specific. RRV was detected in serums from 8 horses for which there were clinical signs consistent with RRV infection such that an acute-phase serum sample was taken and submitted for RRV serology testing. The RRV RT-PCR was analytically sensitive in that it was estimated to detect as little as 50 TCID50 of RRV per mL of serum and was specific in that the primer pairs did not amplify other products from the 8 serum samples. The RRV primers also detected virus in three independent mosquito pools known to contain RRV by virus isolation in cell culture. Samples from horses suspected to be infected with KV and MVEV were not available. Conclusion Despite much anecdotal and serological evidence for infection of horses with RRV actual infection and associated clinical disease are infrequently confirmed. The availability of a specific and analytically sensitive RT-PCR for the detection of RRV provides additional opportunities to confirm the presence of this virus in clinical samples. The RTPCR primers for the diagnosis of KV and MVEV infections were shown to be specific for cell culture grown viruses but the further validation of these tests requires the availability of appropriate clinical samples from infected horses.
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We evaluated the ability of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) to detect West Nile virus (WNV) antibodies in domestic mammals. Sera were collected from experimentally infected horses, cats, and pigs at regular intervals and screened in ELISAs and plaque reduction neutralization tests. The diagnostic efficacies of these techniques were similar.
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Incursions of Japanese encephalitis (JE) virus into northern Queensland are currently monitored using sentinel pigs. However, the maintenance of these pigs is expensive, and because pigs are the major amplifying hosts of the virus, they may contribute to JE transmission. Therefore, we evaluated a mosquito-based detection system to potentially replace the sentinel pigs. Single, inactivated JE-infected Culex annulirostris Skuse and C. sitiens Wiedemann were placed into pools of uninfected mosquitoes that were housed in a Mosquito Magnet Pro (MM) trap set under wet season field conditions in Cairns, Queensland for 0, 7, or 14 d. JE viral RNA was detected (cycling threshold [CT] = 40) in 11/ 12, 10/14, and 2/5 pools containing 200, 1,000, and 5,000 mosquitoes, respectively, using a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR). The ability to detect virus was not affected by the length of time pools were maintained under field conditions, although the CT score tended to increase with field exposure time. Furthermore, JE viral RNA was detected in three pools of 1,000 mosquitoes collected from Badu Island using a MM trap. These results indicated that a mosquito trap system employing self-powered traps, such as the MosquitoMagnet, and a real-time PCR system, could be used to monitor for JE in remote areas.