Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Multiplex”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 793 records · Page 44Linked to original sources
PubMedDate not supplied

Characterization of an analphoid, neocentromere-positive inv dup 8p marker chromosome using multiplex whole chromosome and sub-telomere FISH analyses.

A 30-year-old male patient with mild mental retardation was found to have a small supernumerary marker chromosome (SMC) in 90% of his peripheral blood cells and in 100% of his fibroblast cells. Multiplex whole chromosome and sub-telomere FISH analyses were used to determine that this SMC is an inverted duplicated distal chromosome 8p fragment. Although it was negative for alpha-DNA sequences, this marker had a functional kinetochore (neocentromere) demonstrated by a positive signal with a CENP-C antibody. Apparently intact 8p telomeres at the marker's ends were demonstrated by using a telomere repeat FISH probe. The patient's phenotypically normal mother on G-banding analysis had a small marker chromosome in 8% of her peripheral blood cells in two cultures of the first specimen studied. The marker was not seen in any subsequent maternal peripheral blood or fibroblast specimens. Although it was impossible to further characterize the maternal SMC, it was suggested that the mother had the same marker as the one seen in the proband. Inverted duplicated chromosomal fragments are the most frequent type of analphoid markers. Stable inverted duplicated 8p marker chromosomes were previously reported in three other patients. They all apparently occurred de novo and were found to be positive for kinetochore-associated proteins. Evidence for the possible inheritance of an inverted-duplicated, analphoid SMC was not shown to-date. This study also demonstrates a practical, straightforward approach for analphoid marker characterization in clinical laboratory settings, using whole chromosome multiplex and subtelomere-specific FISH analyses. FISH probes for all sub-telomere chromosomal regions are commercially available and the large majority of analphoid marker chromosomes involve telomere regions.

Adult↗
PubMed2006

Cerebellar volume in offspring from multiplex alcohol dependence families.

BACKGROUND: Increased susceptibility for developing alcohol dependence (AD) might be related to structural differences in brain circuits that influence the salience of rewards and/or modify the efficiency of information processing. The role of the cerebellum in regulating cognitive functions is being increasingly recognized along with its well-known influence on motor performance. Additionally, developmental changes in cerebellar volume during adolescence have been reported. METHODS: Magnetic resonance imaging was used to measure the cerebellum in 17 high-risk adolescent and young adult offspring from multiplex alcohol dependence families and 16 control subjects matched for gender, age, and IQ. RESULTS: High-risk (HR) adolescents/young adults showed increased total cerebellum volume and total grey in comparison with control subjects. Age-related decreases in total grey volume were seen with age, a pattern that was not seen in HR offspring. CONCLUSIONS: Offspring from multiplex families for AD manifest genetic susceptibility by having larger cerebellar volume, which seems to be related to lesser grey matter pruning for age. Larger cerebellar volumes in adult obsessive compulsive disorder (OCD) patients have been reported. This suggests a possible similarity in structural underpinnings for alcohol dependence and OCD.

Adolescent↗
PubMed2006

Screening genetically modified organisms using multiplex-PCR coupled with oligonucleotide microarray.

In this research, we developed a multiplex polymerase chain reaction (multiplex-PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a consecutive reaction to detect a genetically modified organism (GMO). There are a total of 20 probes for detecting a GMO in a DNA microarray which can be classified into three categories according to their purpose: the first for screening GMO from un-transgenic plants based on the common elements such as promoter, reporter and terminator genes; the second for specific gene confirmation based on the target gene sequences such as herbicide-resistance or insect-resistance genes; the third for species-specific genes which the sequences are unique for different plant species. To ensure the reliability of this method, different kinds of positive and negative controls were used in DNA microarray. Commercial GM soybean, maize, rapeseed and cotton were identified by means of this method and further confirmed by PCR analysis and sequencing. The results indicate that this method discriminates between the GMOs very quickly and in a cost-saving and more time efficient way. It can detect more than 95% of currently commercial GMO plants and the limits of detection are 0.5% for soybean and 1% for maize. This method is proved to be a new method for routine analysis of GMOs.

Animals↗
PubMed2006

Visual DNA microarrays for simultaneous detection of Ureaplasma urealyticum and Chlamydia trachomatis coupled with multiplex asymmetrical PCR.

Visual DNA microarrays, based on gold label silver stain (GLSS) and coupled with multiplex asymmetrical PCR, were developed for simultaneous, sensitive and specific detection of Ureaplasma urealyticum and Chlamydia trachomatis. 5'-end-amino-modified oligonucleotides, which were immobilized on glass surface, acted as capturing probes that were designed to bind complementary biotinylated targets DNA. The gold-conjugated streptavidins were introduced to the microarray for specific binding to biotin. The black image of microarray spots, resulting from the precipitation of silver onto nanogold particles bound to streptavidins, were used to detect biotinylated targets DNA visually or with a visible light scanner. Multiplex asymmetrical PCR of U. urealyticum, C. trachomatis and Bacillus subtilis (used as positive control) was performed to prepare abundant biotinylated single-stranded targets DNA, which affected detection efficiency and sensitivity of hybridization on microarray. Plenty of clinical samples of U. urealyticum and C. trachomatis from infected patients were tested using home-made DNA microarrays. For its high sensitivity, good specificity, simplicity, cheapness and speed, the present visual gene-detecting technique has potential applications in clinical fields.

Biosensing Techniques↗
PubMed2004

Multiplex allele-specific PCR assay for differential diagnosis of Hb S, Hb D-Punjab and Hb Tak.

BACKGROUND: Apart from hemoglobin (Hb) E, Hb D-Punjab [beta121(GH4)Glu-Gln] and Hb Tak [beta147Term-Thr] are the two most common beta-chain variants among the Asian population. These two Hb variants have similar alkaline electrophoretic mobilities and HPLC profiles as those of the Hb S [beta6(A3)Glu-Val]. Differential diagnosis of these clinically relevant hemoglobinopathies is therefore problematic. Direct detection of the beta-globin gene mutations would be another diagnostic alternative. METHODS: A simultaneous DNA diagnosis of the three Hb variants was developed based on the multiplex allele-specific polymerase chain reaction (PCR) approach. The method was validated on 10 carriers of Hb D-Punjab, 5 carriers of Hb Tak, 2 carriers of Hb S and 50 normal individuals of Thai origin. RESULTS: The three abnormal Hbs could be correctly diagnosed with the simultaneous PCR approach, and a complete concordance with results using other established methods was obtained. CONCLUSIONS: The multiplex allele-specific PCR approach developed should prove useful in complementing routine Hb analysis for differential diagnosis of these three common Hb variants and should facilitate a program of hemoglobinopathy screening in the region.

Alleles↗
PubMed2005

Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection.

BACKGROUND: As we enter the post-genome sequencing era and begin to sift through the enormous amount of genetic information now available, the need for technologies that allow rapid, cost-effective, high-throughput detection of specific nucleic acid sequences becomes apparent. Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can greatly reduce the time, cost and labor associated with single reaction detection technologies. METHODS: The Luminex xMAP system is a multiplexed microsphere-based suspension array platform capable of analyzing and reporting up to 100 different reactions in a single reaction vessel. This technology provides a new platform for high-throughput nucleic acid detection and is being utilized with increasing frequency. Here we review specific applications of xMAP technology for nucleic acid detection in the areas of single nucleotide polymorphism (SNP) genotyping, genetic disease screening, gene expression profiling, HLA DNA typing and microbial detection. CONCLUSIONS: These studies demonstrate the speed, efficiency and utility of xMAP technology for simultaneous, rapid, sensitive and specific nucleic acid detection, and its capability to meet the current and future requirements of the molecular laboratory for high-throughput nucleic acid detection.

Animals↗
PubMed2004

Multiplex polymerase chain reaction analysis of Glu-1 high-molecular-mass glutenin genes from wheat by capillary electrophoresis with laser-induced fluorescence detection.

The unique bread-making properties of wheat are closely correlated with composition and quantity of high-molecular-mass (HMW) glutenin subunits encoded by the Glu-1 genes. We report the development of a multiplex polymerase chain reaction (PCR) method to identify bread wheat genotypes carrying HMW glutenin allele composition of Glu-1 complex loci (Glu-A1, Glu-B1 and Glu-D1) by capillary electrophoresis(CE) with laser-induced fluorescence (LIF) detection. Two triplex primer sets of HMW glutenin subunit genes were examined. An automated and rapid CE-LIF technique is helpful in the multiplex PCR optimization process. Two fluorescent intercalating dyes (EnhanCE, and YO-PRO-1) are compared for detection of DNA fragments. Amplified DNA fragments of HMW glutenin Glu-1 genes were well separated both by agarose slab-gel electrophoresis and CE, and revealed minor differences between the sequences of 1Ax2*, 1Axnull, 1Bx6, 1Bx7, 1Bx17 and 1Dx5 genes. Moreover, CE technique requires samples of smaller volumes in comparison to slab-gel electrophoresis, and data can be obtained in less than 20 min. There was a very high concordance in the assessment of the molecular size of PCR-generated DNA markers. Fast and accurate identification of molecular markers of Glu-1 genes by CE-LIF can be an efficient alternative to standard procedure separation for early selection of useful wheat genotypes with good bread-making quality.

Base Sequence↗
PubMed2006

Multiplex mapping of CD4 T cell epitopes using class II tetramers.

With the advent of class II tetramer technology, a tetramer-guided epitope mapping (TGEM) technique was developed for the identification of CD4+ T cell epitopes. This allowed the direct identification of epitopes recognized by the responding T cells, which were restricted to the single MHC allele of interest. However, as each individual carries multiple class II alleles, it would be advantageous to design an approach to identify CD4+ epitopes presented by different class II alleles at the same time. In the present study, a multiplex TGEM approach was developed to identify antigenic epitopes presented by multiple HLA class II alleles simultaneously. In this new approach, CD4+ T cells were stained with multiple sets of MHC class II tetramers-each labeled with a unique fluorescent label. Using this multiplex approach, novel epitopes from influenza antigens hemagglutinin and matrix protein presented by multiple class II alleles were identified in a single experimental setting.

Alleles↗
PubMed2003

Quantitative multiplex real-time PCR for the sensitive detection of interferon beta gene induction and viral suppression of interferon beta expression.

Interferon-beta (IFN-beta) protein and activity can be detected by enzyme immunoassays and biological assays. However, precise quantification of low IFN-beta mRNA concentrations, which is advantageous for investigating IFN-beta gene expression in small tissue samples or during the early stage of a virus infection, remains a challenge. Therefore, we established a quantitative real-time PCR (qPCR) for IFN-beta and the housekeeping gene porphobilinogen deanimase (PBGD) in separated assays as well as in a multiplex procedure. Sensitivity for both the templates was less than 20 copies with an intra- and interassay variability of less than 5%. IFN-beta qPCR was utilized to optimize IFN-beta induction with dsRNA polyinosic-polycytidylic acid (poly I:C), delivered by a liposomal transfection agent for reproducible but low, non-cell-toxic IFN-beta concentrations. For studying coxsackievirus B3 (CVB3) interference with IFN-beta expression, CVB3 infected fibroblasts were induced with poly I:C. A significant reduction of IFN-beta mRNA but not PBGD mRNA was demonstrated 5 h after CVB3 infection, indicating a specific inhibition of IFN-beta expression by CVB3 on the mRNA level, in addition to previously reported effects on the translation/post-translation level. In conclusion, sensitive IFN-beta/PBGD multiplex qPCR proved to be a useful tool to study viral interaction with IFN-beta expression.

Gene Expression Regulation↗
PubMed2005

Rapid detection of mecA and nuc genes in staphylococci by real-time multiplex polymerase chain reaction.

A multiplex real-time polymerase chain reaction (RT-PCR) targeting the mecA and nuc genes was developed for the detection of methicillin resistance and identification of Staphylococcus aureus. Novel mecA and nuc primers and fluorescence resonance energy transfer hybridization probes specific for the mecA and nuc genes were evaluated. The assay was performed using the LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) and evaluated against the traditional gel-based multiplex PCR (PCR-gel) method currently used at Royal Perth Hospital. Clinical isolates (n = 222) and isolates from a culture collection library (n = 206) were tested by both assays in parallel. The RT-PCR assay was 100% sensitive and specific for the detection of methicillin resistance and for the identification of S. aureus when compared with the PCR-gel assay. Results from the RT-PCR assay showed 5 isolates with lower efficiency fluorescence curves for the nuc gene PCR fragment. DNA sequencing showed mutations within the region of the probe-binding sites compared with the reference strain. The results of the RT-PCR assay were available within 2 h. This rapid mecA/nuc RT-PCR assay is a suitable and practical tool for the routine detection of methicillin resistance and identification of S. aureus, which can be easily incorporated into the diagnostic molecular microbiology laboratory work flow.

Bacterial Proteins↗
PubMed2006

Multiplex detection of Enterocytozoon bieneusi and Encephalitozoon spp. in fecal samples using real-time PCR.

A multiplex real-time polymerase chain reaction (PCR) method was developed for the simultaneous detection of Enterocytozoon bieneusi (n = 30) and Encephalitozoon spp. (n = 3) in stool samples. The multiplex PCR also included an internal control to detect inhibition of the amplification by fecal constituents in the sample. The assay was performed on species-specific DNA controls (n = 22) and a range of well-defined stool samples (n = 140), and it achieved 100% specificity and sensitivity. The use of this assay in a diagnostic laboratory offers the possibility of introducing DNA detection as a feasible technique in the routine diagnosis of intestinal microsporidian infections.

Animals↗
PubMed2005

Clonorchis sinensis and Opisthorchis viverrini: development of a mitochondrial-based multiplex PCR for their identification and discrimination.

We report a single, one-step PCR approach for detection and discrimination of Clonorchis sinensis and Opisthorchis viverrini in different life-stage forms (adults, metacercariae, and eggs) from fish intermediate hosts and from infected patients. Primers designed for species-specific PCR, amplifying portions of the mitochondrial (mt) genome, were also suitable for a multiplex PCR. The latter was a single, one-step reaction under high stringency conditions, using simultaneously 2 pairs of primers (1 pair for C. sinensis--product size 612 bp, and 1 pair for O. viverrini--product size 1357 bp). Assays using serially diluted templates demonstrated that as little as 0.78 ng of genomic DNA of either species could yield amplicons. Genomic DNA extracted from different life-stage forms including adult worms (of both species), eggs (of O. viverrini), eggs possibly of several trematode species (collected from patients infected with C. sinensis in Vietnam) and mixed metacercariae of common trematodes (collected from fishes in the C. sinensis endemic areas), yielded specific bands of the correct size and their identity was confirmed by sequence analysis. The multiplex PCR approach described here proved to be a species-specific, sensitive and fast tool for accurate diagnosis of clonorchiasis and/or opisthorchiasis, permitting the detection of their metacercariae in infected fishes or adult/eggs from patients in endemic areas.

Animals↗
PubMed2005

Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis.

Bacillus anthracis has four plasmid possible virulence genotypes: pXO1+/pXO2+, pXO1+/pXO2-, pXO1-/pXO2+ or pXO1-/pXO2-. Due to the lack of a specific chromosomal marker for B. anthracis, differentiation of the pXO1-/pXO2- form of B. anthracis from closely related Bacillus cereus group species is difficult. In this study, we evaluate the ability of sspE, pXO1 and pXO2 primers to discriminate individual B. anthracis and the B. cereus group genotypes using multiplex real-time PCR and melting curve analysis. Optimal conditions for successful multiplex assays have been established. Purified DNAs from 38 bacterial strains including 11 strains of B. anthracis and 18 B. cereus group strains were analyzed. Nine of the B. cereus group near-neighbor strains were shown by multilocus sequence typing to be phylogenetically proximate to the B. anthracis clade. We have demonstrated that the four plasmid genotypes of B. anthracis and B. cereus group near-neighbors were differentially and simultaneously discriminated by this assay.

Bacillus anthracis↗
PubMed2006

Species-specific oligonucleotides and multiplex PCR for forensic discrimination of two species of scallops, Placopecten magellanicus and Chlamys islandica.

Characterization of DNA that remains in seafood products after skin, scales, and shells are removed is widely used in forensic species identification, however, ordinary methods may be prohibitively expensive or time-consuming if large sample series need to be discriminated. Forensic discrimination of two species of bivalves commercially harvested from the North Atlantic, sea scallops (Placopecten magellanicus) and Icelandic scallops (Chlamys islandica), was made by means of species-specific oligonucleotides (SSOs) in a multiplex polymerase chain reaction (PCR). The test is a simultaneous in vitro amplification of a portion of the mitochondrial Cytochrome Oxidase I locus with a PCR anchor primer for a sequence identical in both species, and two alternative SSOs that selectively amplify either a 619-bp in Placopecten or a 459-bp DNA fragment in Chlamys. Fragment size and thus species identity are determined directly by gel electrophoresis. In the forensic application, analysis of more than 900 scallops from a series of samples seized from two fishing vessels showed significantly variable proportions of the species from the closed and open fisheries (Placopecten versus Chlamys, respectively). The multiplex SSO test provides a direct means of forensic identification of large population sample series, without the necessity of secondary DNA sequencing, RFLP mapping, or fingerprinting, and can be adapted to other loci and species.

Animals↗
PubMed2004

Application of extended single-reaction multiplex polymerase chain reaction for toxin typing of Staphylococcus aureus isolates in South Korea.

The extended single-reaction multiplex PCR (esr-mPCR) developed in this study to detect staphylococcal enterotoxins (SEs), including SEA, SEB, SEC, SED, SEE, SEH, SEI, and SEJ, requires fewer sets of primers than other conventional multiplex PCRs and can be used to detect newly identified staphylococcal enterotoxins SEs more readily. Esr-mPCR analysis of 141 isolates of Staphylococcus aureus obtained from abattoir and livestock product samples revealed that 27 of the S. aureus isolates were toxigenic, and two were 2 multitoxigenic isolates. The most prevalent SE type was SEI followed by SEA and SEH. In addition, we investigated the clonal relatedness of toxigenic S. aureus isolates by arbitrarily primed PCR (AP-PCR). AP-PCR analysis of toxigenic S. aureus isolates revealed that the discriminatory power of AP-PCR was 9 (D=0.81), 8 (D=0.77), and 10 types (D=0.83) with primers AP1, ERIC2, and AP7, respectively. The combination of three each AP-PCR result could rearrange toxigenic S. aureus isolates into 10 types and five subtypes, with the D-value of 0.92. Interestingly, our data showed that toxigenic S. aureus isolates from different sources had different fingerprinting patterns although some of them carried the same types of SE genes. These data suggest that combinations of esr-mPCR and AP-PCR can provide a powerful approach for epidemiological investigation of toxigenic S. aureus isolates.

Abattoirs↗
PubMed2005

Co-occurrence of boundary and resonance ejection in a multiplexed rectilinear ion trap mass spectrometer.

A method is reported for evaluating ion trap mass analyzers by selection of operating conditions under which both boundary and resonance ejection peaks occur in a single mass scan. The choice of frequency and amplitude of the auxiliary waveform applied for resonance ejection can be such as to produce a resonance ejection mass spectrum with unit resolution or, under selected conditions, signals attributable to both boundary and resonance ejection in a single mass scan. The contrasting mass resolution associated with these two ejection processes is evident in these data. The co-occurrence of the two ejection phenomena is ascribed to the effects of higher-order fields; it is more marked in some rectilinear ion traps (RITs) than in other nominally identical devices, leading to the possibility of using it to compare individual mass analyzers in multiplexed instruments. The method is used to compare multiple ion traps driven by the same RF signal in a fully-multiplexed mass spectrometer, composed of parallel ion source/mass analyzer/detector channels each housed in one quadrant of a specialized vacuum chamber.

Journal Article↗
PubMed2004

High-throughput S-SAP by fluorescent multiplex PCR and capillary electrophoresis in plants.

The inherent replicative mode of transposition endows retrotransposons with considerable advantages as genetic tools in plant genome analysis. Here we present a high-throughput sequence-specific amplification polymorphism (S-SAP) method based on copia-like retrotransposons to fulfill the increasing desire of screening large numbers of samples in plants. Classic approach for digestion, ligation and pre-amplification was combined with optimized fluorescent multiplex PCR for simultaneously selective amplifying S-SAP fragments, and multiple S-SAPs were subsequently detected by capillary electrophoresis using ABI PRISM 3700 capillary instruments. Comparisons of results from multiplex PCR with simplex PCR, and from capillary electrophoresis with slab-gel electrophoresis demonstrated that this method is an efficient, economical, and accurate means for high-throughput and large-scale genotyping retrotransposon variation in plants.

Chromosome Mapping↗
PubMed2006

Multiplex-nested RT-PCR to evaluate latent and lytic Epstein Barr virus gene expression.

This paper describes the development of two multiplex-nested RT-PCR devised to evaluate latent/immortalizing (EBNA1, EBNA2, LMP1 and LMP2) and lytic [immediate early (Zebra), early, and late (VCA), respectively] Epstein Barr virus (EBV) transcripts. Subsequently, the assays have been validated evaluating the EBV latent/lytic gene expression in peripheral blood mononuclear cells (PBMC) from immunocompetent subjects (children with primary EBV infection, past EBV infection and no EBV infection) and from immunosuppressed patients (30 asymptomatic renal transplant recipients and 4 liver transplant patients with diagnosed post-transplant lymphoproliferative disorders [PTLD]). Our two multiplex-nested RT-PCR assays provide a reliable, rapid and sensitive system, enabling the simultaneous detection and identification of seven latent/immortalizing and lytic EBV transcripts. These assays could be employed in further investigations in order to evaluate the EBV transcriptional profile in EBV-related diseases both in immunocompetent and immunocompromised hosts.

Adult↗