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PubMed1999

Multiplex polymerase chain reaction for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples.

AIMS: To develop a multiplex polymerase chain reaction (PCR) for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples. METHODS: Oligonucleotide primers for the amplification of the DNA of these three organisms were optimised for use in combination in the same reaction. PCR products were detected by hybridisation with pooled internal probes using an enzyme linked immunosorbent assay. Those with positive signals were further differentiated using species specific probes. Quality of DNA extraction and PCR inhibition were controlled by amplification of a human mitochondrial gene. A panel of 53 respiratory samples with known results was evaluated blindly. This was followed by a retrospective study on sputa collected from 244 patients with suspected community acquired pneumonia. RESULTS: The multiplex assay had a lower sensitivity than PCR with individual primers by about one log. The resultant sensitivity was considered acceptable for diagnostic use. Of the panel of 53 samples, nine of 11 M pneumoniae, 11 of 11 C pneumoniae, six of seven C psittaci, and 24 of 24 negative samples were correctly identified. Of the 244 patients with pneumonia, seven (2.9%) had detectable M pneumoniae, six (2.5%) had C pneumoniae, and one (0.4%) had C psittaci. The case notes from 11 patients were studied. The PCR finding was of possible significance in at least eight of these patients. CONCLUSIONS: This multiplex PCR assay has the potential to be used as a diagnostic and epidemiological tool. Further prospective studies are needed to establish its clinical value.

Adolescent↗
PubMed2001

Genetic characterisation of invasive breast cancer: a comparison of CGH and PCR based multiplex microsatellite analysis.

AIMS: Comparative genomic hybridisation (CGH) is a reliable tool to gain an overview of all unbalanced chromosomal alterations within a tumour. Nevertheless, the high numbers of tumour cells required and the comparatively low resolution are drawbacks of this technique. Polymerase chain reaction (PCR) based multiplex microsatellite analysis represents a semi-automated, highly reproducible method, which requires small amounts of tumour cells. This is a comparative study of CGH and microsatellite analysis. METHODS: Eighty one samples of invasive breast cancer were investigated by two sensitive multiplex PCRs containing three microsatellites each of six markers (D6S261, D11S907, D6S300, D11S927, D8S272, and D11S925), and two additional microsatellite markers located within intron 1 of the epidermal growth factor receptor gene (egfr) and p53 (p53CA). RESULTS: At least one example of loss of heterozygosity was detectable in all breast cancer tissues. However, the overall rate of accordance between the two methods tested was only 61%. An increasing rate of the number of genetic alterations in each case was mirrored by a constantly increasing fractional allelic loss index. CONCLUSIONS: PCR based multiplex microsatellite analysis using this panel of eight microsatellite markers not only enables the characterisation of cells that have malignant potential in a high frequency of patients with breast cancer, but can also give an estimate of the degree of genetic progression.

Breast Neoplasms↗
PubMed1991

A convenient multiplex PCR system for the detection of dystrophin gene deletions: a comparative analysis with cDNA hybridisation shows mistypings by both methods.

Existing reactions for the multiplex PCR amplification of exons in the dystrophin gene have been modified to produce two multiplex reactions which separately cover the 5' and 3' major deletion 'hotspots' in the gene, and together detect approximately 98% of all deletions detectable by Southern cDNA hybridisation. A comparative study of 148 patients showed mistypings in both the cDNA hybridisation data (4%) and the PCR analysis (1.2%). We suggest means of circumventing the underlying problems in order to avoid mistyping and subsequent misdiagnosis, and conclude that, with appropriate precautions, multiplex PCR amplification can be the method of choice for detecting deletions in the dystrophin gene.

Base Sequence↗
PubMed1992

Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction was developed to simultaneously detect the presence of toxin A and toxin B genes of Clostridium difficile. A 1050-bp fragment of the toxin B gene and a 1217-bp fragment of the toxin A gene were amplified from 42 toxic strains of C. difficile; however, from 10 nontoxic strains the toxin gene fragments were not amplified; these data demonstrate that this multiplex polymerase chain reaction procedure can be used to differentiate between toxic and nontoxic strains. This sensitive and specific multiplex polymerase chain reaction for C. difficile toxins may prove to be a valuable diagnostic procedure.

Bacterial Proteins↗
PubMedDate not supplied

Prenatal diagnosis of arthrogryposis multiplex congenita with increased nuchal translucency but without any underlying fetal neurogenic or myogenic pathology.

Arthrogryposis multiplex congenita is a general term for congenital multiple joint contractures, the aetiology of which is variable. Prenatal diagnosis is usually based on the detection of diminished fetal movements and joint contractures on ultrasound. There are also reports of early diagnosis of arthrogryposis in the first and early second trimester by detection of subcutaneous oedema. We report another case of arthrogryposis multiplex congenita with increased nuchal translucency and scoliosis diagnosed by ultrasonography at 15 weeks of gestation. The pregnancy was terminated at the request of the parents. Post-mortem examination revealed that it was not associated with fetal myopathy or neuropathy. Multiple joint contractures with increased nuchal translucency without any underlying fetal neurogenic and myogenic pathology may be a distinct form of arthrogryposis multiplex congenita.

Arm↗
PubMed1992

Intracranial paramedian hourglass-shaped dermoid associated with hereditary steatocystoma multiplex.

This is the first report to describe the coexistence of two rare diseases, intracranial paramedian hourglass-shaped dermoid and steatocystoma multiplex. A 46-year-old female with a history of steatocystoma multiplex, bradydactylies and kyphosis showed oscillopsia, ataxia and hemifacial spasm. MRI findings suggested a giant dermoid cyst extending from the left middle temporal fossa to the cerebellopontine angle, and this was confirmed surgically. We propose the involvement of some genetic factor or pathological process common to both disorders, steatocystoma multiplex and dermoid. From the clinical point of view, patients with cystic skin lesion should also be checked for intracranial lesion.

Brain Neoplasms↗
PubMed1988

Neurogenic arthrogryposis multiplex congenita: clinical and muscle biopsy findings.

Thirteen cases of arthrogryposis multiplex congenita without evidence of spinal muscular atrophy, congenital muscular dystrophy, or structural myopathy were reviewed. Family history, consanguinity, pregnancy, delivery, number and severity of contractures, and outcome were evaluated. Laboratory investigations had been performed and a biopsy of muscle from an affected limb had been examined histochemically and by electron microscopy. Five biopsies showed fiber type 1 predominance and three had type 2 predominance. Patterns of fiber-type predominance may have resulted from altered neural influence leading to impaired maturation of type 1 or 2 motor units. Nine patients had been followed up for 3-8 years. Two were still not walking at age 8 years. For the 6 who were walking, the mean age at ambulation was 4.7 years. There was no deterioration in power. Muscle biopsy is recommended in arthrogryposis multiplex congenita. Identification of a probable neurogenic arthrogryposis multiplex congenita is important because the condition is not progressive and is apparently not transmitted genetically.

Arthrogryposis↗
PubMed2003

Multiplex nested reverse transcription-polymerase chain reaction for respiratory viruses in acute otitis media.

Because respiratory viruses play an important role in the causation and pathogenesis of acute otitis media (AOM), determining which virus has infected a child is important with respect to vaccines and antiviral drugs. In some instances, this information might be used to prevent the occurrence of AOM. We used a rapid, economical, and sensitive diagnostic system involving a multiplex nested reverse transcription-polymerase chain reaction (RT-PCR) assay to detect various respiratory viruses in clinical specimens of middle ear fluid (MEF) from children with AOM in our hospital. Multiplex RT-PCR was completed on 40 MEF samples from 28 infants and children less than 6 years old with AOM. Viral RNA was detected in 17 MEF samples (43%). Respiratory syncytial virus type A was present in 12 samples, adenovirus in 3, rhinovirus in 2, and influenza A (H3N2) in 1. The multiplex RT-PCR assay is recommended to clinical laboratories that are considering adoption of a molecular technique for viral diagnosis.

Acute Disease↗
PubMed2002

Interscalene brachial plexus block for shoulder surgery in a patient with arthrogryposis multiplex congenita.

A five-year-old child with severe arthrogryposis multiplex congenita and malnutrition underwent surgery for chronic osteomyelitis of the head of the left humerus. The child had typical features of arthrogryposis multiplex congenita, including a difficult airway. Propofol was used for induction and maintenance. Spontaneous respiration was maintained with a nasal airway. Analgesia was provided with an interscalene brachial plexus block placed using a nerve stimulator. No opioid was given. The child had an uneventful recovery with good postoperative analgesia. The anaesthetic implications of arthrogryposis multiplex congenita are discussed.

Arthrogryposis↗
PubMed2001

Long-term survival in a child with arthrogryposis multiplex congenita and spinal muscular atrophy.

Spinal muscular atrophy type 0 is a severe form of spinal muscular atrophy that is usually fatal in the first months of life. These children present with arthrogryposis multiplex congenita and respiratory compromise. We describe a child with spinal muscular atrophy and arthrogryposis multiplex congenita who has had a much better course and is alive without ventilator support at age 6 years. This case illustrates that the prognosis for spinal muscular atrophy and arthrogryposis multiplex congenita cannot always be predicted with certainty.

Arthrogryposis↗
PubMed2003

A dual luciferase multiplexed high-throughput screening platform for protein-protein interactions.

To study the biology of regulators of G-protein signaling (RGS) proteins and to facilitate the identification of small molecule modulators of RGS proteins, the authors recently developed an advanced yeast 2-hybrid (YTH) assay format for GalphaZ and RGS-Z1. Moreover, they describe the development of a multiplexed luciferase-based assay that has been successfully adapted to screen large numbers of small molecule modulators of protein-protein interactions. They generated and evaluated 2 different luciferase reporter gene systems for YTH interactions, a Gal4 responsive firefly luciferase reporter gene and a Gal4 responsive Renilla luciferase reporter gene. Both the firefly and Renilla luciferase reporter genes demonstrated a 40- to 50-fold increase in luminescence in strains expressing interacting YTH fusion proteins versus negative control strains. Because the firefly and Renilla luciferase proteins have different substrate specificity, the assays were multiplexed. The multiplexed luciferase-based YTH platform adds speed, sensitivity, simplicity, quantification, and efficiency to YTH high-throughput applications and therefore greatly facilitates the identification of small molecule modulators of protein-protein interactions as tools or potential leads for drug discovery efforts.

Dose-Response Relationship, Drug↗
PubMed2004

Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics.

BACKGROUND: Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. RESULTS: Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. CONCLUSION: The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.

Journal Article↗
PubMed2001

Multiplex analysis of heterophil antibodies in patients with indeterminate HIV immunoassay results.

We hypothesized that heterophil antibodies reactive with animal proteins used in blot preparation caused nonspecific staining (NSS) on HIV Western blot (WB) studies, causing indeterminate results. We analyzed samples showing NSS on HIV WB using a multiplexed immunoassay to simultaneously measure IgG antibodies to animal IgG (bovine, goat, sheep, mouse) and bovine serum albumin. Heterophil antibodies reactive with IgG from several animal species were detected in 23 (49%) of 47 samples showing NSS on HIV WB; 15 positive samples demonstrated antibodies to all 5 antigens. Similar IgG heterophil antibodies were detected in only 2 (8%) of 24 control samples. Of the HIV WB samples with a positive HIV-1 enzyme-linked immunosorbent assay (ELISA) result at the time of WB testing (11/47), heterophil antibodies were found in 8 (73%) of 11. Preabsorption with bovine, goat, and sheep IgG removed heterophil antibodies detected by the multiplexed assay and, in some cases, eliminated reactivity in ELISA and WB testing. Heterophil antibodies are associated with indeterminate HIV immunoassay results and are an important cause of false-positive HIV ELISA results. Multiplexed immunoassays provide a powerful tool for screening patients for heterophil antibodies and resolving possible false-positive results.

Animals↗
PubMed2002

A multiplexed fluorescent microsphere immunoassay for antibodies to pneumococcal capsular polysaccharides.

We developed a multiplexed indirect immunofluorescent assay for antibodies to pneumococcal polysaccharides (PnPs) based on the Luminex multiple analyte profiling system (Luminex, Austin, TX). The assay simultaneously determines serum IgG concentrations to 14 PnPs serotypes: 1, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 12F; 14, 18C, 19F, and 23F. To assess the specificity of the multiplexed assay for each individual serotype, inhibition-of-binding studies were conducted using adult serum samples obtained after pneumococcal vaccination. Except for the closely related serotypes 9V and 9N, we demonstrated inhibition by homologous serotypes of more than 95% and inhibition by heterologous serotypes of less than 15% for all 14 PnPs serotypes. There was, however, high heterologous inhibition of 50% or greater with some serotypes. These cross-reacting antibodies could not be removed by preabsorption with pneumococcal C-polysaccharide but were removed by additional preabsorption with serotype 22F polysaccharide. The multiplexed Luminex assay showed good overall agreement with a well-established enzyme-linked immunosorbent assay that is currently recommended for evaluation of pneumococcal vaccine immunogenicity.

Antibodies, Bacterial↗
PubMed2003

Superresolution by use of code division multiplexing.

Traditional methods for superresolution have sacrificed field of view for resolution. These methods multiplexed different parts of signals' spectrum on different carriers, and thus managed to transfer a wider range of frequency, in a manner that is similar to frequency division multiplexing in classical communication. We propose code division multiplexing for such an application, which has been shown to have superior capabilities. To enable such mutiplexing we propose a unique setup that creates an incoherent cosine transform of the image. A theoretical analysis of the setup is obtained and later compared with the empirical results.

Journal Article↗
PubMed2003

Invariant pattern recognition by use of a spatial code division multiplexing approach.

Invariant pattern recognition can be achieved by use of harmonic decomposition, for example circular harmonics are used for rotation invariant recognition. A common problem with such methods is that often only a single term of the harmonic decomposition is used, and it does not contain a sufficient amount of the reference energy. Thus discrimination capability is limited, especially in the presence of noise or other disturbances. By using several terms of the harmonic decomposition together this problem can be solved; this can be achieved by the use of code division filter multiplexing. Several harmonic terms are encoded onto a single filter, and the signal is simultaneously correlated with all of them, hence producing enhanced discrimination capabilities. Here two methods are suggested for such encoding. The first involves multiplexing the filters in the Fourier plane, while the second involves multiplexing in the image plane.

Journal Article↗
PubMed2004

Method of hologram multiplexing by use of a fiber bundle with rotary movement.

A simple method of rotating an optical fiber bundle is presented to apply the random-pattern referencing scheme to hologram multiplexing. In the theoretical study, a dependence of the diffraction efficiency on the number of spatial frequencies in the reference pattern is estimated with the Bragg diffraction theory. In the experiment, hologram multiplexing is performed in which 30 holograms are recorded in a LiNbO3 crystal and are read out by rotating a fiber bundle. The result shows that this simple approach enables us to perform the hologram multiplexing and also contributes to the building of a compact optical setup.

Journal Article↗
PubMed2004

Wavelength division multiplexing based on apodized planar holographic Bragg reflectors.

We report wavelength division multiplexing based on lithographically fabricated slab-waveguide-contained planar holographic Bragg reflectors (HBRs). Partial HBR diffractive contour writing and contour displacement are successfully demonstrated to enable precise bandpass engineering of multiplexer transfer functions and make possible compact-footprint devices based on hologram overlay. Four- and eight-channel multiplexers with channel spacings of approximately 50 and approximately 100 GHz, improved sidelobe suppression, and flattop passbands are demonstrated. When a second-order apodization effect, comprising effective waveguide refractive-index variation with written contour fraction, and the effect of hologram overlap on the hologram reflective amplitude are included in the simulation, excellent agreement between predicted and observed spectral passband profiles is obtained. With demonstrated simulation capability, the ability to fabricate general desired passband profiles becomes tractable.

Journal Article↗