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At least 37 records · Page 2Linked to original sources
PubMed2005

Analysis of cellular events using CellCard System in cell-based high-content multiplexed assays.

High-content screening technologies utilize assays that monitor and quantify multiple cellular events. These assays are typically performed on a single cell type with automated microscopy and image analysis. However, in order to better understand the selectivity of a compound across multiple cell lines, these types of assay must be run serially, which is time consuming. The CellCard System developed by Vitra Bioscience enables multiple cell types to be assayed within a single microtiter well, thereby enabling the simultaneous determination of cellular responses across ten cell types. This multiplexed approach could address the demand for assay capacity, increase the quality of the biologic data, reduce timelines, and improve cost-effectiveness in hit identification and lead evaluation. The authors have carried out an in-depth evaluation of this technology platform using ten cancer cell lines and a library of compounds that affect cellular growth through different mechanisms. Multiple assays were used to investigate the compound effects on membrane integrity, cell cycle progression and apoptosis. In this technology review, the authors discuss personal experience with assay validation, data analysis, results such as cell type-specific compound effects, and the potential application of the CellCard System in drug discovery.

Biological Assay↗
PubMed2003

A new quantitative PCR multiplex assay for rapid analysis of chromosome 17p11.2-12 duplications and deletions leading to HMSN/HNPP.

A 1.4-Mb tandem duplication, including the gene for peripheral myelin protein 22 (PMP22) in chromosome 17p11.2-12 is responsible for 70% of the cases of the demyelinating type 1 of Charcot-Marie-Tooth disease or hereditary motor and sensory neuropathy I (CMT1A/HMSN I). A reciprocal deletion of this CMT1A region causes the hereditary neuropathy with liability to pressure palsies (HNPP). The CMT1A duplication increases the PMP22 gene dosage from two to three, the HNPP deletion reduces the gene dosage from two to one. Currently, routine diagnosis of HMSN/HNPP patients is mainly performed with polymorphic markers in-between the repetitive elements flanking the CMT1A region. These show quantitative and/or qualitative changes in case of a CMT1A duplication and a homozygous allele pattern in case of HNPP deletion. In HNPP patients the deletion is usually confirmed by fluorescence in situ hybridisation (FISH). We now developed a reliable, single tube real-time quantitative PCR assay for rapid determination of PMP22 gene dosage directly. This method involves a multiplex reaction using FAM labelled Taqman-probe with TAMRA quencher derived from PMP22 exon 3 and a VIC labelled probe with non-fluorescent quencher from exon 12 of the albumin gene as internal reference. Copy number of the PMP22 gene was determined by the comparative threshold cycle method (deltadeltaCt). Each sample was run in quadruplicate and analysed at two different threshold levels. The level giving the smallest standard deviation was scored. We evaluated this method through the retrospective analysis of 252 HMSN patients with known genotype and could confirm the previous findings in 99% of cases. Two patients were wrongly diagnosed with microsatellite analysis while quantitative real-time PCR identified the correct genotype, as confirmed by FISH. Thus, this method shows superior sensitivity to microsatellite analysis and has the additional advantage of being a fast and uniform assay for quantitative analysis of both CMT1A and HNPP.

Biological Assay↗
PubMed2005

Two-color multiplex assay for the identification of orthopox viruses with real-time LUX- PCR.

The LUX [Light Upon eXtension] is a real-time detection system that can be used for the detection and quantification of pathogens nucleic acids. In this study we used a universal LUX approach, a variation of the LUX detection system, for identifying Orthopoxvirus nucleic acids in real time. This approach enables the design of sequence-specific primer sets in high identity genome sequences. The assay described here is designed to allow simultaneous detection of Variola and other orthopox viruses in a multiplex format, with a limit of detection in the range of 50--100 copies of the Orthopoxvirus genome. Regression analysis showed that the assay was linear over seven orders of magnitude, with 0.97 correlation coefficient. The sensitivity and specificity of the assay, as determined from a panel of 100 samples that contained nucleic acids from a variety of bacteria and viral species, were rated at 98%. Thus, the assay offers a sensitive and specific tool for simultaneous identification and quantification of Variola and other orthopox viruses, and the approach allows more flexible sequence-specific primers design for pox viruses as well as other microbial pathogens.

Animals↗
PubMed2001

Automated multiplex assay system for simultaneous detection of hepatitis B virus DNA, hepatitis C virus RNA, and human immunodeficiency virus type 1 RNA.

We have developed an automated multiplex system for simultaneously screening hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) in blood donations. The assay, designated AMPLINAT MPX HBV/HCV/HIV-1 Test (AMPLINAT MPX), consists of virus extraction and target sequence-specific probe capture on specimen preparation workstation GT-X (Roche Diagnostics K.K., Tokyo, Japan) and amplification and detection by TaqMan PCR on the ABI PRISM 7700 Analyzer (Perkin-Elmer Applied Biosystems, Foster City, Calif.). An internal control (IC) is incorporated in the assay to monitor the extraction, target amplification, and detection processes. The assay yields qualitative results without discrimination of the three targets. Detection limits (95% confidence interval) are 22 to 60 copies/ml for HBV, 61 to 112 IU/ml for HCV, and 33 to 66 copies/ml for HIV-1, using a specimen input volume of 0.2 ml. The AMPLINAT MPX assay detects a broad range of genotypes or subtypes for all three viruses and has a specificity of 99.6% for all three viruses with seronegative specimens. In an evaluation of seroconversion panels, the AMPLINAT MPX assay detects HBV infection an average of 24 days before the detection of HBsAg by enzyme immunoassay. HCV RNA was detected an average of 31 days before HCV antibody. HIV-1 RNA was detected an average of 14 days before HIV-1 antibody and an average of 9 days before p24 antigen. The Japanese Red Cross has been evaluating the AMPLINAT MPX system since October 1999. The clinical performance indicates that the AMPLINAT MPX system is robust, sensitive, and reproducible, with a high percentage of valid assay runs (96.8%), a low false-positive rate (0.34%), and a low IC failure rate (0.24%).

Blood Donors↗
PubMed2005

Three multiplex assays for detection of Borrelia burgdorferi sensu lato and Borrelia miyamotoi sensu lato in field-collected Ixodes nymphs in North America.

Two hundred fifty New Jersey field-collected Ixodes scapularis Say ticks and 17 Colorado Ixodes spinipalpis Hadwen & Nuttall ticks were tested using three separate multiplex real-time polymerase chain reaction (PCR) assays. One assay targets the rrs-rrlA IGS region of Borrelia spp. to detect Borrelia burgdorferi sensu lato (s.l.) and Borrelia miyamotoi s.l. The second assay targets the ospA region of B. burgdorferi s.l. to detect B. burgdorferi sensu stricto (s.s.), Borrelia bissettii, and Borrelia andersonii. The final assay targets the glpQ region of B. miyamotoi s.l. to differentiate B. miyamotoi LB-2001 and Borrelia lonestari. A testing scheme combining these tests yielded 18% of tested I. scapularis ticks surveyed from New Jersey positive for B. burgdorferi s.s., 3.2% I. scapularis ticks positive for B. miyamotoi LB-2001, and 41.2% I. spinipalpis ticks positive for B. bissettii surveyed from Colorado.

Animals↗
PubMed2003

Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways.

The current interest in expression of groups of functionally related genes creates a demand for novel experimental tools. We describe a multiplex ligation-dependent amplification procedure (RT-MLPA), which accurately quantifies up to 45 transcripts of interest in a one-tube assay. The output, a set of fluorescent DNA fragments, is analysed via capillary sequencer and spreadsheet software. The procedure is highly sensitive and reproducible over a 100-fold range of input RNA, with excellent compatibility with RT-PCR and microarrays. We targeted two comprehensive sets of human genes: 35 apoptosis regulators and 30 genes involved in inflammation. Both probe sets accurately assessed specific changes in gene expression in two relevant model systems. Stimulation of lymphocytes with various Toll-like receptor (TLR) ligands induced distinct inflammatory profiles. Furthermore, osteosarcoma cells treated with cytostatic drugs showed as primary response strong up-regulation of the apoptogenic p53-inducible PUMA transcript. Suppression by RNAi validated that indeed Puma expression was responsible for apoptosis induction. Thus, RT-MLPA enables relevant changes in transcription patterns to be quickly pinpointed and guide further experiments. This can be an advantage compared to hypothesis-free whole genome screens where large numbers of differentially expressed genes can obscure functional interpretation.

Apoptosis↗
PubMed2005

Quantitative multiplex assay for simultaneous detection and identification of Indiana and New Jersey serotypes of vesicular stomatitis virus.

In order to establish a rapid and reliable system for the detection of vesicular stomatitis virus (VSV), we developed a quantitative reverse transcription-PCR assay for the detection, quantification, and differentiation of the major serotypes, VSV Indiana and VSV New Jersey, using a closed-tube multiplex format. The detection system is based on the recently invented primer-probe energy transfer (PriProET) system. A region of the gene encoding the RNA-dependent RNA polymerase was amplified by using VSV-specific primers in the presence of two serotype-specific fluorescent probes. By incorporating nucleotide analogues in the primers, both serotypes were amplified with similar efficiencies. The generation of specific amplicons resulted in fluorescent signals for either of the two serotypes, and the specificities of the reactions were confirmed from the melting temperature profiles of the fluorescent probes. The limits of detection were found to be less than 10 50% tissue culture infective doses/ml for both serotypes. The diagnostic value of the new method was tested with clinical materials from experimentally infected pigs, and it is concluded that the method is a powerful tool for the rapid identification of VSV.

Animals↗
PubMed2004

Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening.

BACKGROUND: Newborn screening for deficiency in the lysosomal enzymes that cause Fabry, Gaucher, Krabbe, Niemann-Pick A/B, and Pompe diseases is warranted because treatment for these syndromes is now available or anticipated in the near feature. We describe a multiplex screening method for all five lysosomal enzymes that uses newborn-screening cards containing dried blood spots as the enzyme source. METHODS: We used a cassette of substrates and internal standards to directly quantify the enzymatic activities, and tandem mass spectrometry for enzymatic product detection. Rehydrated dried blood spots were incubated with the enzyme substrates. We used liquid-liquid extraction followed by solid-phase extraction with silica gel to remove buffer components. Acarbose served as inhibitor of an interfering acid alpha-glucosidase present in neutrophils, which allowed the lysosomal enzyme implicated in Pompe disease to be selectively analyzed. RESULTS: We analyzed dried blood spots from 5 patients with Gaucher, 5 with Niemann-Pick A/B, 11 with Pompe, 5 with Fabry, and 12 with Krabbe disease, and in all cases the enzyme activities were below the minimum activities measured in a collection of heterozygous carriers and healthy noncarrier individuals. The enzyme activities measured in 5-9 heterozygous carriers were approximately one-half those measured with 15-32 healthy individuals, but there was partial overlap of each condition between the data sets for carriers and healthy individuals. CONCLUSION: For all five diseases, the affected individuals were detected. The assay can be readily automated, and the anticipated reagent and supply costs are well within the budget limits of newborn-screening centers.

Blood Specimen Collection↗
PubMed2006

Preparation of silica encapsulated quantum dot encoded beads for multiplex assay and its properties.

Novel -COOH modified polystyrene beads were prepared by sulfonation grafting, and the surface area and pore volume are greatly improved in comparison with the swelling-treated beads. The optimization coating time is 4 h, and the corresponding -COOH content is approximately 2.1 mmol/g. The scanning electron microscope results show that the silica particles deposited on the beads and formed a silica shell that decreases the leakage of quantum dots (QDs) preferably and improves the bar code stability greatly. The anti-photobleaching of silica-coated beads was studied systemically, and the results show that the half-decay time (t1/2) of the coated beads increases to 537 s--seven times longer than that of the uncoated ones. Further DNA probe hybridization experiments indicated that the coding signal and target signal can be detected simultaneously and that the assays based on these probe-conjugated silica/QD/polystyrene beads have good specificity and sensitivity that can detect a concentration as low as 0.01 microg/ml target DNA in denatured calf thymus DNA solution, indicating that it is feasible to use this kind of bead for multiplex analysis.

DNA↗
PubMed2004

A single multiplex assay to identify major malaria vectors within the African Anopheles funestus and the Oriental An. minimus groups.

The African Anopheles funestus and the Oriental An. minimus groups are closely related and composed of major malaria vectors in Africa and Southeast Asia, respectively. None of the species of either the An. funestus or the An. minimus group can be identified with absolute certainty using the adult morphology. Polymorphisms present on the internal transcribed spacer 2 (ITS2) of ribosomal DNA allowed the development of 10 primers that combined with an universal forward primer lead to a simple and sensitive multiplex allele-specific polymerase chain reaction (AS-PCR). Moreover, the possible additional amplification of the entire ITS2 allows one to detect other anopheline species in sympatry with members of both groups not included in this assay and serves as a control band. This universal PCR method permits the discrimination of 10 species within the subgenus Cellia, among which figure three major malaria vectors, and constitutes a very efficient and powerful tool to improve our knowledge on these species distribution and biology. Not only restricted to anophelines, this AS-PCR could also be developed and applied to other insect groups.

Africa↗
PubMed2006

A highly informative, multiplexed assay for the indirect detection of hemophilia A using five-linked microsatellites.

BACKGROUND: Hemophilia A is a severe bleeding disorder caused by almost 1000 different known mutations in the F8C gene. Direct mutation analysis is sometimes difficult for this disorder. When a mutation cannot be found, linkage analysis can be used for prenatal and carrier diagnosis. AIM: To develop a rapid and effective system for carrier detection and prenatal diagnosis of hemophilia A based on a single-multiplexed polymerase chain reaction (PCR) reaction utilizing five microsatellite markers. PATIENTS AND METHODS: Two intronic microsatellites and three other markers flanking the factor VIII gene were ascertained, and primers were designed for multiplex PCR amplification. A kindred with Hemophilia A was tested for linkage using the panel of primers, and informativity in the general population was ascertained by testing 50 unrelated females. RESULTS: Co-amplification of all microsatellites was optimized using DNA extracted by standard methods. Rapid detection and sizing of products were carried out using an automated DNA sequencer. The combined microsatellite panel was informative in each of the kindreds tested, and in 100% of the 50 unrelated females (95% CI 94.2-100%). CONCLUSIONS: This method enables the indirect detection of hemophilia A for patients in whom mutations cannot be found, facilitating carrier testing and prenatal analysis. It is rapid and straightforward compared with many other published protocols, and offers a high degree of informativity.

Base Sequence↗
PubMed2006

A multiplex assay with 52 single nucleotide polymorphisms for human identification.

A total of 52 SNPs reported to be polymorphic in European, Asian and African populations were selected. Of these, 42 were from the distal regions of each autosome (except chromosome 19). Nearly all selected SNPs were located at least 100 kb distant from known genes and commonly used STRs. We established a highly sensitive and reproducible SNP-typing method with amplification of all 52 DNA fragments in one PCR reaction followed by detection of the SNPs with two single base extension reactions analysed using CE. The amplicons ranged from 59 to 115 bp in length. Complete SNP profiles were obtained from 500 pg DNA. The 52 loci were efficiently amplified from degraded samples where previously only partial STR profiles had been obtained. A total of 700 individuals from Denmark, Greenland, Somalia, Turkey, China, Germany, Taiwan, Thailand and Japan were typed, and the allele frequencies estimated. All 52 SNPs were polymorphic in the three major population groups. The mean match probability was at least 5.0 x 10(-19) in the populations studied. Typical paternity indices ranged from 336 000 in Asians to 549 000 in Europeans. Details of the 52 SNP loci and population data generated in this work are freely available at http://www.snpforid.org.

DNA Fingerprinting↗
PubMedDate not supplied

Simultaneous analysis of six microsatellite markers in Atlantic cod ( Gadus morhua): a novel multiplex assay system for use in selective breeding studies.

A novel hexaplex assay system including Gmo8, Gmo19, Gmo35, Gmo37, Tch11, and Tch12 microsatellites from Atlantic cod, consisting of trinucleotide or tetranucleotide repeat units, is introduced. All 6 loci were coamplified in a single reaction employing dye-labeled primers. Alleles from these loci were sized using an internal standard by automated sample processing in an ABI 310 Genetic Analyser. Amplified alleles in profiles containing selected microsatellites were typed clearly, providing easily interpretable results. Sequencing data indicated that alleles at all loci consisted of simple repeat units. This may help minimize the likelihood of stuttering upon polymerase chain reaction amplification. The results suggest that the presented hexaplex assay system may be a useful tool in a selective breeding program in which genetic identification will allow different genotypes to be reared together from fertilization. This should have a great impact as it will make selective breeding more efficient.

Animal Husbandry↗
PubMed2004

Cytometric bead array: a multiplexed assay platform with applications in various areas of biology.

The introduction of flow cytometric bead-based technology has added a new approach for investigators to simultaneously measure multiple analytes in biological and environmental samples. This new technology allows for (1) evaluation of multiple analytes in a single sample; (2) utilization of minimal sample volumes to glean data; (3) reproducibility and results comparative with previous experiments; (4) direct comparison with existing assays; and (5) a more rapid evaluation of multiple samples in a single platform. The cytometric bead array (CBA) system enables simultaneous measurement of multiple analytes in sample volumes too small for traditional immunoassays. Results have been presented for the analysis of a variety of human cytokines. In addition, the technology allows for the design and creation of assays to measure a variety of analytes including inflammatory mediators, chemokines, immunoglobulin isotypes, intracellular signaling molecules, apoptotic mediators, adhesion molecules, and antibodies. New initiatives put forward by the Human Genome Project and the FDA require the development and use of assays for the rapid simultaneous quantitation of multiple analytes. The CBA technology provides the ability to quantify multiple proteins within a given sample, with precision and consistency.

Anaphylatoxins↗
PubMed2002

Multiplex assay for detection of strain-specific antibodies against the two variable regions of the G protein of respiratory syncytial virus.

The role of strain differences in respiratory syncytial virus (RSV) disease has not been clearly defined. To investigate the possibility that strain differences contribute to susceptibility to repeat infections, we developed assays to detect antibodies to the two variable regions of the RSV G protein by cloning and expressing the internal variable region at amino acids (aa) 60 to 172 (g1) and the carboxy-terminal variable region at aa 193 to the carboxy terminus (g2) from different genotypes of RSV. The purified proteins were covalently linked to beads with different proportions of red and orange fluorescent dyes and reacted against serum specimens. Antibody reacting against the differently colored beads, and thus against different G polypeptides, was detected by use of flow cytometry and the Luminex system. This assay system detected group- and, to some extent, genotype-specific responses to RSV infection and can be used to investigate the role of strain differences in RSV disease.

Animals↗
PubMed2006

Technical validation of a TM Biosciences Luminex-based multiplex assay for detecting the American College of Medical Genetics recommended cystic fibrosis mutation panel.

The American College of Medical Genetics (ACMG) and the American College of Obstetrics and Gynecology have recommended population-based carrier screening for cystic fibrosis to include 23 mutations and 5 polymorphisms in the cystic fibrosis transmembrane regulator gene(CFTR). We estimate 20% of all pregnant women are being tested for their CF carrier status. We assessed two commercially available analyte-specific reagents (ASRs) capable of testing all 25 mutations of the original ACMG-recommended panel, Tag-It CFTR40 + 4 Luminex-based reagent from Tm Biosciences, and our current assay platform, CF Genotyper V. 3.0 from Abbott/Celera. Blinded testing using genomic controls containing known CFTRmutations demonstrated that the Tag-It platform detected all mutations on the ACMG-recommended panel. We next performed a platform comparison with 1,029 consecutive patient samples. There were no discrepant results in 1,029 consecutive analyses between the two platforms, yielding an impressive figure of >25,000 individual genotypes without error for both platforms. In conclusion, both the Abbott/Celera ASR reagent and the Luminex-based Tag-It CF ASR reagent are appropriate for use in the clinical laboratory.

Alleles↗