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At least 1,531 records · Page 85Linked to original sources
PubMed2007

Comparison of GenoType MTBC with RFLP-PCR and multiplex PCR to identify Mycobacterium tuberculosis complex species.

The aims of the work presented here were to evaluate a commercial system based on DNA hybridization technology on nitrocellulose strips (GenoType MTBC; Hain Diagnostika, Nehren, Germany) for identifying Mycobacterium tuberculosis complex isolates to the species level and to compare the results with those obtained using the RFLP-PCR and multiplex PCR molecular techniques, biochemical tests and susceptibility testing. The commercial system correctly identified 102 of 103 (99.03%) strains studied, indicating it is capable of discriminating among the tuberculosis complex species.

DNA Probes↗
PubMed2005

Identification of Mytilus spp. and Pecten maximus in Irish waters by standard PCR of the 18S rDNA gene and multiplex PCR of the 16S rDNA gene.

Two molecular protocols for the identification of mussel and scallop have been developed using specific primers targeting the mitochondrial 16S ribosomal DNA gene and the nuclear 18S ribosomal DNA gene. Primers for the mitochondrial 16S ribosomal DNA gene in multiplex polymerase chain reaction (PCR) protocols yielded diagnostic DNA fragments for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis (335 bp), the king scallop Pecten maximus (382 bp) and the black scallop Mimachlamys varia (398 bp). DNA from the queen scallop Aequipecten opercularis showed no consistent PCR amplification of the 16S rDNA gene. Primers for the nuclear 18S rDNA gene in standard PCR protocols yielded similar-sized, diagnostic DNA fragments (approx. 190 bp) for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis, the king scallop Pecten maximus, the black scallop Mimachlamys varia, and the queen scallop Aequipecten opercularis. Both protocols have been tested with Mytilus spp., P. maximus, and 6 other bivalve species from a wide range of locations in Irish and European waters. Cross reaction of the specific primers with DNA template from any of the 6 other bivalve species was not observed. Rapid DNA extraction using FTA Card technology and the16S rDNA primers allowed for the detection of at least 10 mussel larvae in a subsample of natural plankton.

Animals↗
PubMed2006

Identification of the ETT2 locus in human diarrheagenic Escherichia coli by multiplex PCR.

An Escherichia coli type III secretion system 2 (ETT2) locus was discovered in enterohemorrhagic E. coli O157:H7. To determine presence or absence of the ETT2 locus in diarrheagenic E. coli, a multiplex polymerase chain reaction (PCR) encoding Shiga toxins (stx1 and stx2), intimin (eaeA), and ETT2 (etrA) was developed for rapid detection. The ETT2 locus was identified not only in Shiga toxin-producing E. coli (STEC) but also in various non-STEC.

Diarrhea↗
PubMedDate not supplied

Simultaneous evaluation of circulating chemokine and cytokine profiles in elderly subjects by multiplex technology: relationship with zinc status.

Cellular components of both adaptive and innate immune systems produce different chemokines and cytokines, involved in different signalling pathways among cells, and modulate effector function during immune response, playing a key role in the regulation of the type and extent of the immune response in the elderly. We evaluated the circulating concentration of selected chemokines: MCP-1, MIP-1alpha, IL-8, RANTES together with IL-6 and TNF-alpha in plasma obtained from a group of healthy old subjects, in order to highlight possible differences in the synthesis of these factors, assuming that both the cytokine and the chemokine networks are remodelled with ageing. The simultaneous evaluation was performed by a multiplex analysis system. In addition, since micronutrient deficiency may underlie an inflammatory response, the association between chemokine levels and a nutritional element such as zinc was also evaluated, since the immune system is the first system to be affected by changing zinc levels, due to its high cell turnover. A progressive age-related increase of plasma concentrations of all soluble factors was observed. The increment was particularly evident for IL-6, IL-8, MCP-1 and TNF-alpha in the over 85-year-old group in concomitance with increasing percentages of subjects with low circulating levels of zinc. In conclusion, the remodelling of chemokine profiles, skewed to Th2 response by both advanced ages and circulating levels of zinc, might reflect different states of activation and/or responsiveness of the human immune cell/mediator network, thus influencing the ability to develop rapid innate and long-lasting adaptive immune responses with advancing age.

Age Distribution↗
PubMed2005

Multiplex cytokine profiling of initial therapeutic response in patients with chronic hepatitis C virus infection.

Currently available prognostic tools are inadequate to discern the molecular basis of the heterogenic response in hepatitis C virus (HCV)-infected patients treated with the current standard of therapy. The expression and biological function of immune mediators have been shown to be critical in all phases of the immune response to HCV infection and likely therefore influence host response. Herein, a biometric multiplex serum cytokine assay was utilized to characterize the immunomodulatory effects of host response in 10 HCV patients. Serum levels of 17 cytokines were compared before and after 1 month of treatment and against controls. Overall serum cytokine levels were significantly higher in patients (P < 0.05) than controls. Additionally, viral titers decreased in all patients after 1 month of therapy, as did overall serum cytokine levels in the cohort (P < 0.05). To assess relationships between changes in cytokine levels and changes in viral titer, the cohort was divided into three statistically distinct subgroups based on changes in viral titers. Specific sets of cytokines decreased in each group: decreases in CCL4, interleukin (IL)-2, CXCL8, and IL-1beta correlated with the greatest drops in viral titer, decreases in IL-5, granulocyte colony stimulating factor (G-CSF), and CCL4 correlated with moderate drops in viral titer, and only CCL2 correlated with the lowest drops in viral titer. Interestingly, decreases in CCL4 levels correlated with decreases in viral titers in all patients. CCL4 controls leukocyte influx and thus propagates inflammation. In conclusion, these data raise the possibility that characteristic changes in host response modulate the therapeutic response, demonstrating the prognostic power of serum cytokine profiling in chronic HCV.

Adult↗
PubMed2005

Epicocconone, a new cell-permeable long Stokes' shift fluorescent stain for live cell imaging and multiplexing.

Epicocconone is a heterocyclic natural product from the fungus Epicoccum nigrum that fluoresces weakly in the green (520 nm). However, cells exposed to epicocconone rapidly absorb the dye and become bright orange fluorescent because the natural product reacts reversibly with proteins. The orange fluorescence is enhanced in lipophilic environments, allowing the visualization of membranous organelles and lipid rafts but does not stain oligonucleotides. As the unconjugated dye has no orange fluorescence, there is no need to wash out the excess fluorophore. Epicocconone is a neutral, non-toxic, small molecule that appears to diffuse readily into live of fixed cells without the need for permeabilization. These features enable the real-time imaging of live cells and the study of organelle movements. Cells stained with epicocconone are excitable by common lasers (UV, 405, 488, and 532 nm) and its long Stokes' shift allows multiplexing applications with more common short Stokes' fluorophores using a single light source.

Benzopyrans↗
PubMed2006

One-step multiplex RT-PCR assay for the detection of peste des petits ruminants virus in clinical samples.

A single-tube one-step multiplex RT-PCR was standardized to amplify both 337 bp and 191 bp fragments of N and M genes of peste des petits ruminants virus (PPRV), respectively, and only a 337 bp fragment of N gene of Rinderpest virus (RPV). The RT-PCR using purified viral RNA was easily adopted for direct detection of PPRV in clinical field samples and its differentiation from RPV. The amplified N and M gene products were confirmed to be PPRV- and RPV-specific by their size in 1.5% agarose gel and restriction analysis. In the assay, the Qiagen one-step RT-PCR kit containing the Ominiscript and Sensiscript reverse transcriptases and Hot star Taq DNA polymerase was utilized. The sensitivity of the assay was found to be 100 fg of PPRV RNA. Compared with a two-step assay, the one-step assay is easier and time-saving as it requires just a single buffer for both reactions, reverse transcription (RT) and PCR. In experimentally infected goats, PPRV was detectable by the one-step RT-PCR in nasal and ocular swabs 7-17 days post infection (p.i.). and in oral swabs 7-15 days p.i. Out of 32 clinical field samples tested, 18 were positive by sandwich ELISA (S-ELISA), while 22 were positive by the one-step RT-PCR.

Animals↗
PubMed2006

Rapid detection of avian influenza virus a and subtype H5N1 by single step multiplex reverse transcription-polymerase chain reaction.

Outbreaks of H5N1 highly pathogenic avian influenza (HPAI) virus caused great economic losses to the poultry industry and resulted in human deaths in Thailand and Viet Nam in 2004. Rapid typing and subtyping of H5N1 viruses, especially from clinical specimens, are desirable for taking prompt control measures to prevent the spread of the disease. Here, we developed a set of oligonucleotide primers able to detect, type and subtype H5 and N1 influenza viruses in a single step multiplex reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from allantoic fluid or from specimens with guanidinium isothiocyanate reagent. Reverse transcription and PCR were carried out with a mixture of primers specific for influenza viruses of type A, subtype H5 and N1 in a single reaction system under identical conditions. The amplified DNA fragments were analyzed by agarose gel electrophoresis. All the H5N1 viruses tested in the study and the experimental specimens presented three specific bands by the method established here. The results presented here suggest that the method described below is rapid and specific and, therefore, could be valuable in the rapid detection of H5N1 influenza viruses in clinics.

Animals↗
PubMed1991

Cardiac myocytes and dendritic cells harbor human immunodeficiency virus in infected patients with and without cardiac dysfunction: detection by multiplex, nested, polymerase chain reaction in individually microdissected cells from right ventricular endomyocardial biopsy tissue.

Two hundred fifteen patients infected with human immunodeficiency virus (HIV) participated in a prospective longitudinal study of HIV-related heart disease. Evaluation included signal-averaged electrocardiography and echocardiography. Fifteen patients underwent endomyocardial biopsy, 5 had cardiovascular symptoms and 10 did not. Cardiac myocytes or dendritic cells were prepared by individual cell microdissection to sort them from other cell types such as interstitial cells or circulating blood elements. HIV proviral sequences were amplified in samples of 15 to 20 cells of each type by multiplex, nested, polymerase chain reaction and hybridized to 32P-labeled probes specific for regions within the gag and pol genes of HIV-1. The results showed the presence of HIV sequences in myocytes of 2 of 5 patients with cardiac symptoms and in 6 of 10 without. Thus, symptomatic HIV cardiomyopathy did not appear to be a direct consequence of the virus on myocardial cells. In dendritic cells, HIV sequences were detected in 5 of 5 patients with cardiac symptoms and in 8 of 10 with apparently normal ventricular function. Furthermore, dendritic cells were somewhat more numerous in the myocardium of symptomatic than asymptomatic patients. Our studies are the first to directly detect the HIV genome in purified cardiac myocytes from patients with and without cardiac dysfunction. Our findings do not support a direct role of the virus in myocardial dysfunction. However, the results do suggest that the interstitial dendritic cells may be involved in some manner in the development of cardiac dysfunction observed in HIV-infected patients.

Adult↗
PubMed1978

Neuropathic form of arthrogryposis multiplex congenita. Report of 3 cases with complete necropsy, including the first reported case of agenesis of muscle spindles.

In 3 infants with arthrogryposis multiplex congenital (AMC) complete necropsy, including removal of the entire spinal cord, was performed. Histologically, spinal type (neurogenic) atrophy of skeletal muscles in conjunction with spinal motor neurone depletion, unaccompanied by noticeable gliosis, were the most preminent features common to all cases. In addition to these, one infant (Case 1) showed total absence of muscle spindles as evident from the examination of several hundred step-serial paraffin sections covering 11 different levels in 36 samples taken from a wide range of skeletal muscles of both upper and lower extremities. This is believed to be the first published case of agenesis of muscle spindles.

Arthrogryposis↗
PubMed1988

Arthrogryposis multiplex congenita of the temporomandibular joint.

A case of arthrogryposis multiplex congenita involving the temporomandibular joint is reported. This is only the second reported case in which surgery was used in an attempt to alleviate the limited mandibular opening that may be associated with this uncommon condition. The clinical findings at the time of surgery differed significantly from those classically associated with other joints afflicted with this condition, suggesting that a different pathophysiologic mechanism may be involved.

Adult↗
PubMed1992

Sex determination and milk protein genotyping of preimplantation stage bovine embryos using multiplex PCR.

A method for determining the sex and milk protein genotypes (RFLPs) of preimplantation stage bovine embryos using multiplex polymerase chain reaction (PCR) is described. Day 6 to 7 embryos were micromanipulated to isolate 5 to 6 cells. These cells were then dried in reaction tubes for transport to the laboratory. Subsequently, two sets of PCRs were performed using Y chromosome, k-casein and beta-lactoglobulin gene specific primers, followed by electrophoretic analysis of the PCR products. The presence or absence of the Y chromosome was ascertained in 90 of 92 embryos. Moreover, the k-casein specific fragment was amplified and detected in all these embryos. The PCR products were digested in order to genotype the k-casein gene. In 70% of the embryos, the beta-lactoglobulin specific fragment was amplified, although together with some unspecific fragments.

Journal Article↗
PubMed1984

Computer-controlled temperature multiplexer for thermal dosimetry studies.

Thermistor probes are widely used for mapping thermal distributions in hyperthermia research. To facilitate multi-probe mapping, a computer-controlled multiplexer for automated thermistor impedance measurement and temperature conversion is described. A complete hardware interface schematic is shown. An algorithm which calculates individual temperature values as a function of thermistor impedance is tested. Total error is quantified by calculating the absolute error between the theoretical temperatures obtained from the algorithm and the experimental calibration temperatures obtained from a National Bureau of Standards referenced mercury thermometer. If calibration intervals of 1.00 degree C or less are used, the total error of the system is less than 0.10 degree C.

Biomedical Engineering↗
PubMed1993

A functional basis for the association of HLA class II genes and susceptibility to multiple sclerosis: cellular immune responses to myelin basic protein in a multiplex family.

This study has examined the cellular response to myelin basic protein (MBP) in a multiplex family with multiple sclerosis (MS). A total of 81 MBP-specific T cell lines (TCLs) were derived from three affected siblings and four healthy siblings. No difference was observed in estimated precursor frequencies of MBP-specific TCLs or peptide specificity of TCLs when comparing affected and unaffected siblings. MBP-specific TCLs from affected siblings, however, were restricted to the DRw15/DQw6 allele more frequently than those from unaffected siblings (P < 0.02). These data suggest that restriction of autoantigen-specific T cells may be the functional basis for disease susceptibility related to HLA class II inheritance.

Adult↗
PubMed1993

A novel candidate autoantigen in a multiplex family with multiple sclerosis: prevalence of T-lymphocytes specific for an MBP epitope unique to myelination.

Although the major isoform of myelin basic protein (MBP) in the healthy adult CNS is the 18.5-kDa protein, other isoforms containing exon 2 encoded protein (21.5 kDa and 20.2 kDa) exist and are expressed primarily during myelin formation. Since remyelination is a prominent feature in MS lesions, we examined the frequencies of T cell lines (TCLs) specific for epitopes within exon 2 encoded MBP (X2MBP), and also within 18.5-kDa MBP, in members of a multiplex family with MS. TCLs specific for X2MBP were as prevalent as TCLs specific for immunodominant epitopes within 18.5-kDa MBP. In addition, while frequencies of TCLs specific for 18.5-kDa MBP were no different between the affected and unaffected, the frequency of X2MBP-specific TCLs correlated with disease.

Aged↗
PubMed1988

Arthrogryposis multiplex congenita: otolaryngologic diagnosis and management.

Arthrogryposis multiplex congenita (AMC) is an uncommon congenital disorder characterized by multiple fixed joint deformities and non-progressive neuromuscular dysfunction. A small fraction of these infants will present with otolaryngologic problems resulting from cranial nerve weakness, muscle dysplasia, or structural dysharmony of the head and neck. The charts of 50 patients with AMC were reviewed to determine the incidence of these findings. A summary of the literature is presented discussing the etiology, pathophysiology, diagnosis and management of this interesting clinical problem.

Arthrogryposis↗
PubMed1995

Multiplex polymerase chain reaction for human herpesvirus-6, human cytomegalovirus, and human beta-globin DNA.

Human cytomegalovirus and human herpesvirus-6 are closely related viruses which cause similar diseases, have similar cellular repositories of latent infection, and may be detected largely in the same types of clinical specimens. DNA amplification appears likely to play an increasing role in the diagnosis of recent and remote infection with these agents. A sensitive multiplex polymerase chain reaction was therefore developed for the two viruses and for human beta-globin DNA. Optimization of parameters such as the primers, primer concentrations, magnesium concentration, and buffer constituents was crucial in achieving a sensitive assay. Preliminary results indicated that the assay could simultaneously monitor DNA extraction from clinical specimens and allow detection of HCMV or HHV-6 in patients with diseases possibly caused by either pathogen.

Base Sequence↗
PubMed1996

Multiplex polymerase chain reaction for adenovirus and herpes simplex virus in eye swabs.

Adenoviruses and herpes simplex virus (HSV) can cause clinically indistinguishable episodes of acute eye disease. Adenovirus infection is associated with nosocomial outbreaks and HSV may result in episodes of recurrent ocular inflammation. In a comparison of multiplex PCR for the two viral DNAs and virus isolation in cell culture, identical results were obtained for 18 of 20 specimens (positive for adenovirus in 5, HSV in 5, and negative in 8). One specimen was falsely negative for each viral DNA. Inclusion of human beta-globin primers in the adenovirus-HSV reaction was precluded by a consequential 10--100-fold reduction in sensitivity for the two viral targets and by the failure of beta-globin DNA amplification at the annealing temperature (45 degrees C) required to ensure detection of adenoviruses of serotypes 7 and 11 with the selected adenovirus primers. A single-target beta-globin PCR gave positive results with 19 of the 20 specimens prepared by treatment with proteinase K lysis buffer, indicating the effectiveness of this simple DNA extraction procedure. Nonetheless, the availability of effective antiviral therapy for HSV made monitoring for extraction failure using human primers crucial to avoid false-negative results for HSV DNA. Adenovirus-HSV PCR has considerable potential for the rapid diagnosis of viral eye disease particularly if beta-globin primers can be included in the reaction.

Adenovirus Infections, Human↗