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 991 records · Page 55Linked to original sources
PubMed2004

Multiplex real-time RT-PCR for prospective evaluation of WT1 and fusion gene transcripts in newly diagnosed de novo acute myeloid leukemia.

Prognostic assessment is crucial for the management of AML. Although the use of karyotype analysis for risk-stratification is widely accepted, prognosis of AML remains ambiguous, particularly for patients categorized into the intermediate cytogenetic risk group and additional markers are required for an accurate prediction of outcome. For this study, we used multiplex real-time RT-PCR, which can simultaneously quantify WT1 and 10 distinct fusion gene transcripts, to prospectively evaluate the pre-treatment bone marrow findings of 53 de novo AML patients. Five patients with normal karyotype or insufficient metaphases detected by conventional karyotype analysis proved to have AML1-MTG8, CBFbeta-MYH11 or PML-RARalpha fusion transcripts. WT1 overexpression was observed in 92% of the patients, and the levels were significantly higher in the cytogenetic favorable risk group, especially patients with PML-RARalpha. WT1 levels also correlated with the percentage of blasts in bone marrow, especially in cases of core-binding factor leukemia. There was no association between initial WT1 levels and outcome in terms of event-free survival or overall survival. These results suggest that multiplex real-time RT-PCR is rapid and useful for the precise cytogenetic stratification of AML, and that WT1 levels at presentation correlate with several biologic features of leukemia, but have no prognostic significance.

Adolescent↗
PubMed1998

A multiplex RT-PCR assay for analysis of relative transcript levels of different members of multigene families: application to Arabidopsis calmodulin gene family.

The high degree of conservation of nucleotide sequences among different members of a multigene family poses problems in analysis of expression patterns governed by each member of the gene family. In this report we describe a simple, semi-quantitative and single tube multiplex RT-PCR assay for simultaneous and relative expression analysis with an application to all the six members of Arabidopsis calmodulin multigene family. In the multiplex primer set, individual gene specific primers were derived from 3'-untranslated region of the genes and a single common primer from the conserved exonic region. Transcriptional activation of all the members of the calmodulin gene family in response to touch was monitored. The results demonstrate that two of the genes are not regulated by touch; however, the other four that are induced by touch show a differential response including their kinetics of induction.

Arabidopsis↗
PubMed2005

The homogeneous multiplexed system--a new method for autoantibody profile in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multi-systemic autoimmune disease leading to immunological aberrations and excessive multiple autoantibody production. The aim of this study was to investigate the prevalence of multiple autoantibodies in SLE patients utilizing the multiplex system method. We analyzed the presence of elevated titers of anti-Ro, anti-La, anti-RNP, anti-Sm, anti-Jo1, anti-centromere, anti-Scl-70, anti-histone, and anti-dsDNA antibodies in 199 serum samples (113 SLE patients, 86 healthy donors). We compared the type, level and number of autoantibodies and the correlation between the autoantibody profile and disease severity utilizing the SLEDAI score. Elevated titers of at least one autoantibody were detected in 48% of 42 SLE patients. Elevated titers of anti-Ro antibodies were most commonly detected. The distribution of specific autoantibodies was: anti-Ro- 23.8%, anti-dsDNA- 19%, antihistone- 19%, anti-RNP- 14.2%, anti-La antibodies- 11.9%, anti-Sm- 7.1%, anti-Scl 70-4.7%, and anti-centromere- 2.4%. Utilizing ROC analysis, the sensitivity and specificity of anti-DNA antibodies at a cutoff value of 34 IU/ml were 87.1% and 79.4% respectively. Elevated titers of anti-Jo1 antibody were not detected. There was a correlation with the titer of anti-Ro antibodies and disease activity by the SLEDAI score. Seven patients harbored one autoantibody only, 15 patients harbored 2-3 autoantibodies, 3 patients harbored 4-5 autoantibodies, and one patient harbored 6 autoantibodies. A correlation between the number of autoantibodies per patient and disease severity was found. One patient with a multitude of autoantibodies had severe lupus and a myriad of clinical manifestations. In conclusion, the multiplex system is specific and sensitive, provides an autoantibody profile in a single test, and may be useful as a diagnostic test for SLE. Elevated anti-Ro antibodies are associated with severe disease. An autoantibody load may be indicative of more severe disease.

Antibodies, Antinuclear↗
PubMed2003

Common and unique susceptibility loci in Graves and Hashimoto diseases: results of whole-genome screening in a data set of 102 multiplex families.

The autoimmune thyroid diseases (AITDs), comprising Graves disease (GD) and Hashimoto thyroiditis (HT), develop as a result of a complex interaction between predisposing genes and environmental triggers. Previously, we identified six loci that showed evidence for linkage with AITD in a data set of 56 multiplex families. The goals of the present study were to replicate/reject the previously identified loci before fine mapping and sequencing the candidate genes in these regions. We performed a whole-genome linkage study in an expanded data set of 102 multiplex families with AITD (540 individuals), through use of 400 microsatellite markers. Seven loci showed evidence for linkage to AITD. Three loci, on chromosomes 6p, 8q, and 10q, showed evidence for linkage with both GD and HT (maximum multipoint heterogeneity LOD scores [HLOD] 2.0, 3.5, and 4.1, respectively). Three loci showed evidence for linkage with GD: on 7q (HLOD 2.3), 14q (HLOD 2.1), and 20q (LOD 3.3, in a subset of the families). One locus on 12q showed evidence of linkage with HT, giving an HLOD of 3.4. Comparison with the results obtained in the original data set showed that the 20q (GD-2) and 12q (HT-2) loci continued to show evidence for linkage in the expanded data set; the 6p and 14q loci were located within the same region as the previously identified 6p and 14q loci (AITD-1 and GD-1, respectively), but the Xq (GD-3) and 13q (HT-1) loci were not replicated in the expanded data set. These results demonstrated that multiple genes may predispose to GD and HT and that some may be common to both diseases and some are unique. The loci that continue to show evidence for linkage in the expanded data set represent serious candidate regions for gene identification.

Chromosome Mapping↗
PubMed1985

Data acquisition from a multiplex, quadrupole mass spectrometer.

Although various methods have been used to correct the output of a respiratory mass spectrometer for the delay and rise time in its response, thereby reducing the error in measured gas concentrations, there are additional considerations in the case of a multiplex mass spectrometer. The measured signal from the detector is a series of discrete samples of the concentration of several different gases rather than a continuous monitor of a single gas concentration as generated by other types of mass spectrometer. If the time constant of the mass spectrometer is of the same order as the interval between samples of a gas in the multiplex mode, correction techniques based on continuous-time analysis would not be as valid as those based on discrete-time analysis. Such correction techniques were compared to the use of a simple time shift. For multibreath gas washout analysis with simulated worst-case 'square wave breathing' it was found that, because of the complex nature of the response of the mass spectrometer, a simple time shift provided a reduction in error nearly equal to that of an additional first order response correction, and that such further corrections may be unnecessary or even invalid under some circumstances.

Humans↗
PubMed2006

Design, in vitro and in vivo assessment of a multi-channel sieve electrode with integrated multiplexer.

This paper reports on the design, in vitro and in vivo investigation of a flexible, lightweight, polyimide based implantable sieve electrode with a hybrid assembly of multiplexers and polymer encapsulation. The integration of multiplexers enables us to connect a large number of electrodes on the sieve using few input connections. The implant assembly of the sieve electrode with the electronic circuitry was verified by impedance measurement. The 27 platinum electrodes of the sieve were coated with platinum black to reduce the electrode impedance. The impedance magnitude of the electrode sites on the sieve (geometric surface area 2,200 microm(2)) was |Z(f=1kHz)| = 5.7 kOmega. The sieve electrodes, encased in silicone, have been implanted in the transected sciatic nerve of rats. Initial experiments showed that axons regenerated through the holes of the sieve and reinnervated distal target organs. Nerve signals were recorded in preliminary tests after 3-7 months post-implantation.

Action Potentials↗
PubMed2001

Multiplex sequencing by hybridization.

One of the limitations of classical sequencing by hybridization (SBH) is the inefficient use of probes in the "all k-mers" array. This limitation occurs due to the relatively short length (roughly the square root of C) of target that may be reconstructed by an array with C probes. We propose a new strategy, multiplex sequencing by hybridization, that greatly increases the efficiency of target reconstruction. In the typical multiplex SBH method, many different target sequences are simultaneously reconstructed (as compared to a single sequence in classic SBH). This is accomplished by pooling the target sequences and performing several hybridization experiments. This procedure makes more efficient use of probes so that the combined length of sequence reconstructed per DNA array increases significantly as compared to classical SBH.

Algorithms↗
PubMed2001

High-level multiplex DNA amplification.

We present data on efficient amplification of large number of DNA targets using a single-tube polymerase chain reaction (PCR). This is a further enhancement of our approach to multiplexed PCR based on PCR suppression, which allows multiple DNA amplification using only one sequence-specific primer per amplicon while the second primer is common for all targets (Broude, N.E., et al., Proc. Natl. Acad. Sci. USA 98, 206-211, 2001). The reaction conditions have been optimized for simultaneous synthesis of 30 DNA targets, mostly consisting of fragments containing single nucleotide polymorphisms (SNP). The size of the amplified fragments, derived from many different human chromosomes, varies from 100 to 600 bp. We conclude that this method has potential for highly multiplexed DNA amplification useful for SNP analyses, DNA diagnostics, and forensics.

Base Sequence↗
PubMed2003

A rapid and definitive test for Charcot-Marie-Tooth 1A and hereditary neuropathy with liability to pressure palsies using multiplexed real-time PCR.

Alterations in gene copy number have been shown to cause disease in humans. Two of the most common inherited peripheral neuropathies, Charcot-Marie-Tooth 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), are two such diseases resulting from alteration in gene copy number of the dosage sensitive peripheral myelin protein 22 (PMP22) gene. Many complicated and laborious diagnostic tests exist for the diagnosis of these diseases. The aim of our study was to develop the first quantitative multiplex real-time PCR assay for the diagnosis of CMT1A and HNPP. A total of 160 individuals who were known to have CMT1A, HNPP, or were normal from previous testing were assayed by our multiplex real-time PCR method. The results confirmed the previously determined gene copy number of all patient and control individuals tested. The range of ratio values between the disease and control groups were easily defined. The assay is accurate, simple, and cost effective and can detect a 50% change in gene copy number. This represents an ideal assay for any small diagnostic laboratory.

Case-Control Studies↗
PubMed2003

Multiplex ligation-dependent probe amplification (MLPA) detects large deletions in the MECP2 gene of Swedish Rett syndrome patients.

Mutations in the methyl-CpG-binding protein-2 (MECP2) gene on Xq28 have been found to be a cause of Rett syndrome (RS). In a previous mutation screening, we found MECP2 mutations in 81% of Swedish classical Rett women. In this study, we have analyzed 22 patients for MECP2 deletions using multiplex-ligation-dependent probe amplification (MLPA). Clinically, 11 of the patients who were classical Rett women, 3 were forme fruste, 1 was congenital RS, and 7 were Rett variants. As inclusion criteria, we used DNA from patients in whom previous sequencing results showed no mutations in coding portions of the MECP2 gene. MLPA is a method based on multiplex PCR. In one PCR, as many as 40 probes are amplified with the same primers. The specificity of the amplification products is determined by the site-specific hybridization of each probe construct, prior to amplification. Each PCR product has a unique length, which makes it possible to identify it by size separation. In 3 of 11 (27%) classical Rett women, we detected large deletions in MECP2 using MLPA. All these patients had deletions covering two exons; in 2 cases the deletion involved exons 3 and 4 and, in one case, exons 1 and 2 were missing. In the forme fruste, congenital and Rett-variant patients, we found no large deletions. We have found that MLPA is useful when it comes to finding large deletions compromising whole exons in MECP2. Used as a complementary method to DNA sequencing, it revealed new MECP2 mutations in classical RS patients.

Chromosomal Proteins, Non-Histone↗
PubMed2002

Multiplexed screening assay for mRNA combining nuclease protection with luminescent array detection.

The principles and performance are described for the ArrayPlate mRNA assay, a multiplexed mRNA assay for high-throughput and high-content screening and drug development. THP-1 monocytes grown and subjected to compound treatments in 96-well plates were subjected to a multiplexed nuclease protection assay in situ. The nuclease protection assay destroyed all cell-derived mRNA, but left intact stoichiometric amounts of 16 target-specific oligonucleotide probes. Upon transfer of processed cell lysates to a microplate that contained a 16-element oligonucleotide array at the bottom of each well, the various probe species were separated by immobilization at predefined elements of the array. Quantitative detection of array-bound probes was by enzyme-mediated chemiluminescence. A high-resolution charge-coupled device imager was used for the simultaneous readout of all 1536 array elements in a 96-well plate. For the measurement of 16 genes in samples of 25000 cells, the average standard deviation from well to well within a plate was 8.6% of signal intensity and was 10.8% from plate to plate. Assay response was linear and reproducibility was constant for all detected genes in samples ranging from 1000 to 50000 cells. When THP-1 monocytes were differentiated with phorbol ester and subsequently activated with bacterial lipopolysaccharide that contained different concentrations of dexamethasone, dose-dependent effects of dexamethasone on the mRNA levels of several genes were observed.

Anti-Inflammatory Agents↗
PubMed2003

An encoded particle array tool for multiplex bioassays.

The detection of both gene function at the genomic scale and protein assays at a proteomic scale is developing into an essential tool in the discovery and development of new medicines. A wide variety of techniques have been used that allow the analyst to assess the information gathered from a SNP up to a fully developed immunoassay. Despite these advances, there remains an ongoing drive to keep the cost of research and development down to reduce the cost of bringing a new drug or diagnostic to market. The demand to carry out large-scale bioassay analysis using yet smaller reagent volume and at a very low cost per test is gaining much importance as seen in the growing need to perform multiplex bioassays. In this article, we describe an analytical system that is being developed to address such a need for multiplexing bioassays at a low, affordable cost providing a flexible alternative consumable to the current technologies. This novel system utilizes inexpensive fabricated polymeric material produced by standard MEMS technology, which allows the attachment, through a variety of coupling chemistries, of oligonucleotides, proteins, antibodies, and antigens. These can then be analyzed by simple scanning, with either a flow cytometer or a simple optical reader, providing a robust, sensitive microarray assay system.

Oligonucleotide Array Sequence Analysis↗
PubMed2004

Problems in isolation of Campylobacter jejuni from frozen-stored raw milk and bovine fecal samples: genetic confirmation using multiplex PCR.

The objectives of this study were to evaluate the use of various protocols for the isolation of Campylobacter jejuni from bulk tank milk and bovine fecal samples that were stored frozen for varying times, and to develop a rapid DNA-based protocol that distinguishes C. jejuni from other thermophilic Campylobacter spp. The pathogen was recovered from fecal samples that had been stored for 96-251 days at -20 degrees C with glycerol as the cryopreservative. In a separate study, C. jejuni-positive bovine fecal samples were stored at 5 degrees C for up to 70 days without compromising subsequent recovery of the pathogen. However, the pathogen could not be recovered from pathogenpositive fecal samples stored with or without glycerol (5 mL/11 g sample) for 21 days at -20 degrees C. Bolton broth (BB) and Bolton broth with 5% blood (BBB), containing BB supplement, were used for enrichment. Bacterial isolation was achieved by streaking from BB and BBB, and filtering from BBB onto blood-free charcoal cefoperazone deoxycholate agar (CCDA). The use of BB for the recovery of Campylobacter was more sensitive than BBB, and streaking achieved better isolation rates than filtration. Multiplex PCR incorporating thermophilic Campylobacter-specific 23S rRNA and C. jejuni-specific hippuricase gene sequences was used to confirm C. jejuni. All 265 bulk tank milk samples analyzed were negative for C. jejuni, whereas five of 411 (1.2%) fecal samples tested positive. This is the first report that has used a combination of sequences of the two genes in a multiplex format to identify C. jejuni to the species level. The method described has potential for routine use in the detection of thermophilic Campylobacter in farm environmental samples as well as other samples.

Animals↗
PubMed2006

Development and evaluation of a real-time FRET probe based multiplex PCR assay for the detection of prohibited meat and bone meal in cattle feed and feed ingredients.

A novel real-time fluorescent multiplex polymerase chain reaction (PCR) assay for detecting and discriminating between bovine, ovine, and caprine contaminates in cattle feed was developed that simultaneously performs quality control monitoring on both the DNA extraction process and the level of PCR inhibition in the final DNA extract in a single PCR run. The assay used a single set of primers and two sets of FRET probes targeting the ruminant-specific mitochondrial cytochrome b gene. An internal control PCR reaction targeting a region of the chloroplast RNA polymerase beta-subunit (rpobeta) gene, which is conserved among plants, was incorporated into the ruminant multiplex PCR reaction in order to both monitor the DNA extraction method and to test for the presence of PCR inhibitors. The detection limit for bovine and ovine contaminates was evaluated over a period of two sets of six trials on 15 different types of cattle feed and feed ingredients spiked with known concentrations of bovine meat and bone meal (BMBM) and lamb meat and bone meal (LMBM). The assay was able to detect 0.05% w/w BMBM contamination and 0.1% w/w LMBM contamination in all samples of cattle feed and feed ingredients tested.

Animal Feed↗
PubMed1998

A multiplex ARMS test for 10 cystic fibrosis (CF) mutations: evaluation in a prenatal CF screening program.

In Maine, prenatal screening for cystic fibrosis (CF) is offered through primary care providers. Cheekbrush (buccal) samples are routinely tested for eight mutations by multiplex PCR amplification of five exons, followed by dot-blot hybridization with pooled allele-specific oligonucleotides (ASO). The ASO methodology is widely used and effective, but somewhat time and labor intensive when applied to CF carrier testing or couple-based prenatal screening in the general pregnant population. Amplification Refractory Mutation System (ARMS) is an improvement of the PCR that allows rapid detection of mutations involving single base changes or small deletions/insertions. In this study, two multiplex ARMS reactions are used to test for 10 common CF mutations. Clinical evaluation of the ARMS test includes a retrospective study of 140 banked samples (54 cell line, proficiency testing, and buccal controls; 86 clinical buccal samples) with known CF genotype (57 with CF mutations, 83 no mutation), followed by a prospective trial in which 309 buccal samples are analyzed con-currently using both methods. The success rate of the ARMS test in buccal lysates is comparable to the ASO method; all CF mutations are successfully identified. For testing nonsterile buccal lysates with low DNA concentrations, optimized performance in the ARMS method is obtained using Amplitaq Gold polymerase. The ARMS method developed is easy, rapid (1 day), and avoids the need for ASO probe labeling, dot-blotting and autoradiography. This study provides further evidence that ARMS methodology is suitable for clinical CF mutation analysis.

Cheek↗
PubMed2004

Improved molecular diagnosis of dystrophin gene mutations using the multiplex ligation-dependent probe amplification method.

Mutation detection in the DMD gene defective in Duchenne (DMD) and Becker muscular dystrophies (BMD) is complicated by the presence of 79 exons. The majority of recognized mutations are, however, copy number changes of individual exons, which traditionally have been identified by three common multiplex polymerase chain reaction (PCR) assays and/or Southern blotting. Here we report the use of the newly developed quantitative assay multiplex ligation-dependent probe amplification (MLPA) to determine the copy number of each of the 79 DMD exons in 182 males and 14 carrier females referred to our diagnostic facility on the clinical suspicion of DMD or BMD. The MLPA method confirmed all previously recognized mutations and identified an additional 28, including four point mutations. Also, the assay reliably identified 7 carrier females, which are usually not easily recognized. In our hands the method is highly reproducible, easy to handle, and has increased our mutation pick-up rate by a total of 33%.

DNA Mutational Analysis↗
PubMed2006

A fluorescent multiplex-DGGE screening test for mutations in the BRCA1 gene.

Screening for mutations in the BRCA1 gene is challenging because of the wide spectrum of mutations found in this large gene. As the extensive exon 11 is commonly screened by the protein truncation test (PTT), here a fluorescent multiplex denaturing gradient gel electrophoresis (FMD) mutation screening technique was developed to test the remaining numerous small exons and splice sites of the gene. The method is based upon the use of an efficient multiplex polymerase chain reaction (PCR) amplification of the target regions, followed by denaturing gradient gel electrophoresis (DGGE) separation of the amplicon mixture, and the immediate achievement of results by wet gel scanning. The technique was applied to screen 16 samples with different BRCA1 sequence variants distributed over 12 exons. All variants were detected. In addition, 188 DNA samples from ovarian cancer patients were screened, identifying 22 new sequence variants (11.7% of the samples) and 243 common polymorphisms in the BRCA1 locus. Variants included 16 single nucleotide substitutions, 3 deletions of 2 nucleotides, 1 deletion of 4 nucleotides, and 2 insertions of 1 nucleotide. The FMD test provides an accurate, fast, nonradioactive and cost-efficient way to scan the BRCA1 gene with high sensitivity and an ease of result interpretation. This technique may prove to be a useful research tool for the detection of mutations and polymorphisms in the BRCA1 gene and for large-scale epidemiologic studies.

BRCA1 Protein↗
PubMed1997

Identification of methicillin-resistant staphylococci by multiplex polymerase chain reaction assay.

A multiplex polymerase chain reaction (PCR) assay using oligonucleotide primers to detect mecA and 16S ribosomal RNA gene was developed to aid in identification of methicillin-resistant staphylococci. Validation included 99 isolates of staphylococcus grouped into one of five categories: methicillin-susceptible coagulase-negative staphylococcus (MSCNS), methicillin-resistant coagulase-negative staphylococcus (MRCNS), methicillin-susceptible Staphylococcus aureus (MSSA), high beta-lactamase producing S aureus (HiBSA), and methicillin-resistant S aureus (MRSA). mecA was detected in MRSA (21/21), and in MRCNS (20/20), but not in MSSA (0/20). mecA was occasionally detected in HiBSA (1/19) and MSCNS (3/19). This multiplex PCR assay was also used to test 30 clinical isolates of coagulase-negative staphylococci with discrepancies between results of in vitro tests for susceptibility to oxacillin and was found to be valuable when a more definitive determination of intrinsic methicillin-resistance was desired.

DNA, Bacterial↗