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At least 1,153 records · Page 64Linked to original sources
PubMed2006

Harnessing asymmetrical substrate recognition by thermostable EndoV to achieve balanced linear amplification in multiplexed SNP typing.

Multiplexed amplification of specific DNA sequences, by PCR or by strand-displacement amplification, is an intrinsically biased process. The relative abundance of amplified DNA can be altered significantly from the original representation and, in extreme cases, allele dropout can occur. In this paper, we present a method of linear amplification of DNA that relies on the cooperative, sequence-dependent functioning of the DNA mismatch-repair enzyme endonuclease V (EndoV) from Thermotoga maritima (Tma) and Bacillus stearothermophilus (Bst) DNA polymerase. Tma EndoV can nick one strand of unmodified duplex DNA, allowing extension by Bst polymerase. By controlling the bases surrounding a mismatch and the mismatch itself, the efficiency of nicking by EndoV and extension by Bst polymerase can be controlled. The method currently allows 100-fold multiplexed amplification of target molecules to be performed isothermally, with an average change of <1.3-fold in their original representation. Because only a single primer is necessary, primer artefacts and nonspecific amplification products are minimized.

Base Pair Mismatch↗
PubMed2004

Multiplex PCR detection of clinical and environmental strains of Vibrio vulnificus in shellfish.

In this study, we developed a PCR-based rapid detection method for clinically important pathogenic strains of Vibrio vulnificus. Positive amplification of the 504-bp viuB fragment was seen in all 22 clinical isolates tested but only in 8 out of 33 environmental isolates. The combination of the species-specific 205-bp vvh fragment along with viuB in a multiplexed PCR enabled us to confirm the presence of potentially pathogenic strains of V. vulnificus. No amplification of other Vibrio spp. or non-Vibrio bacteria was evidenced, suggesting a high specificity of detection by this method. The sensitivity of detection for both targeted genes was 10 pg of purified DNA, which correlated with 10(3) V. vulnificus CFU in 1 mL of pure culture or 1 g un-enriched seeded oyster tissue homogenate. This sensitivity was improved to 1 CFU per gram of oyster tissue homogenate in overnight-enriched samples. A SYBR Green I based real-time PCR method was also developed that was shown to produce results consistent with the conventional PCR method. Application of the multiplexed real-time PCR to natural oyster tissue homogenates exhibited positive detection of vvh in 51% of the samples collected primarily during the summer months; however, only 15% of vvh positive samples exhibited viuB amplicons. The rapid, sensitive, and specific detection of clinically important pathogenic V. vulnificus in shellfish would be beneficial in reducing illnesses and deaths caused by this pathogen.

Amides↗
PubMed2004

Multiplexing schemes for generic SNP genotyping assays.

A generic genotyping assay utilizes a fixed set of reagents, which is independent of the actual target sample, to determine all present alleles. An example is the interrogation of several amplicons spanning polymorphic sites using an all k-mer array. Due to the high cost associated with a genotyping experiment, it is desirable to design a set of experiments, which maximizes the number of SNPs that can be genotyped in parallel per assay. In this study we investigate algorithmic approaches for optimally multiplexing SNP genotyping using generic assays. We devise a graph theoretic formulation of the problem and use it to derive an approximation algorithm for the problem, and several practical heuristics. We apply our methods to simulated and real data, for evaluating the multiplexing rates afforded by generic techniques. The results on real human data show the practicality of generic approaches for genotyping, allowing, e.g., the genotyping of 5000 SNPs using four all 7-mer arrays.

Algorithms↗
PubMed1991

Steatocystoma multiplex: appearance at mammography.

The authors present mammographic findings in a patient with steatocystoma multiplex. Numerous oil cysts were seen in both breasts at mammography. The mammographic appearance is characteristic of, if not pathognomonic for, steatocystoma multiplex.

Breast Diseases↗
PubMed2007

Early detection of head and neck cancer: development of a novel screening tool using multiplexed immunobead-based biomarker profiling.

Squamous cell carcinoma of the head and neck (SCCHN) is an aggressive disease that has been linked to altered immune, inflammatory, and angiogenesis responses. A better understanding of these aberrant responses might improve early detection and prognosis of SCCHN and provide novel therapeutic targets. Previous studies examined the role of multiplexed serum biomarkers in small cohorts or SCCHN sera. We hypothesized that an expanded panel comprised of multiple cytokines, chemokines, growth factors, and other tumor markers, which individually may show some promising correlation with disease status, might provide higher diagnostic power if used in combination. Thus, we evaluated a novel multianalyte LabMAP profiling technology that allows simultaneous measurement of multiple serum biomarkers. Concentrations of 60 cytokines, growth factors, and tumor antigens were measured in the sera of 116 SCCHN patients before treatment (active disease group), 103 patients who were successfully treated (no evidence of disease group), and 117 smoker controls without evidence of cancer. The multimarker panel offering the highest diagnostic power was comprised of 25 biomarkers, including epidermal growth factor, epidermal growth factor receptor, interleukin (IL)-8, tissue plasminogen activator inhibitor-1, alpha-fetoprotein, matrix metalloproteinase-2, matrix metalloproteinase-3, IFN-alpha, IFN-gamma, IFN-inducible protein-10, regulated on activation, normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein-1alpha, IL-7, IL-17, IL-1 receptor-alpha, IL-2 receptor, granulocyte colony-stimulating factor, mesothelin, insulin-like growth factor binding protein 1, E-selectin, cytokeratin-19, vascular cell adhesion molecule, and cancer antigen-125. Statistical analysis using an ADE algorithm resulted in a sensitivity of 84.5%, specificity of 98%, and 92% of patients in the active disease group correctly classified from a cross-validation serum set. The data presented show that simultaneous testing using a multiplexed panel of serum biomarkers may present a promising new approach for the early detection of head and neck cancer.

Adult↗
PubMed1998

Mononeuropathy multiplex in hemophilia: an electrophysiologic assessment.

Single nerve lesion or mononeuropathy caused by extrinsic compression of hematoma or hemarthrosis is consistently reported for hemophilic patients. To study the manifestation of peripheral nerve lesions, an extensive nerve conduction study of peripheral nerves and specific nerves in correlation with clinical involvement was carried out on 126 hemophilic patients. We found 24 patients with 34 peripheral nerve lesions. The frequency of nerve lesion positively correlated with the severity of hemophilia (p < 0.01) and increasing age (p < 0.05). Nerve lesions occurred more commonly in lower extremities than in upper extremities. The femoral nerve was most commonly involved. Seven patients were identified to have mononeuropathy multiplex with 2 or more nerve lesions involving more than one extremity. All of these patients had chronic condition. Our findings suggest that mononeuropathy multiplex may occur in hemophilic patients.

Adolescent↗
PubMed2006

Evaluation of serotypes of Streptococcus pneumoniae isolated from otitis media patients by multiplex polymerase chain reaction.

The increasing difficulty in the management of pneumococcal acute otitis media in parallel with increases in antimicrobial-resistant strains has led to much interest in pneumococcal capsular types for the adoption of effective prevention by vaccines. This study shows that multiplex polymerase chain reaction is a valuable and expeditious method for the capsular typing of pneumococci. The multiplex polymerase chain reaction method accurately detects the majority of serotypes and serogroups frequently isolated from pediatric patients with acute otitis media, allowing the characterization of the colonization patterns for further implications of pneumococcal vaccines.

Bacterial Capsules↗
PubMed1997

Expression of five selected human mismatch repair genes simultaneously detected in normal and cancer cell lines by a nonradioactive multiplex reverse transcription-polymerase chain reaction.

Abnormalities in at least 1 of 5 mismatch repair (MMR) genes (hMSH2, hMLH1, hPMS1, hPMS2 and GTBP/hMSH6) are found in hereditary nonpolyposis colon cancer and sporadic colon cancers. We used a single-reaction multiplex reverse transcription (RT)-polymerase chain reaction (PCR), with the beta-actin gene as an internal control, to simultaneously evaluate expression of these 5 known human MMR genes in normal and tumor cell lines with known or uncharacterized mutations in MMR genes. The relative quantitation of the transcripts is demonstrated by controlling the number of PCR cycles and titrating cDNA with a dose-curve. The 13 normal cell lines tested were derived from normal lymphocytes, skin, thymus, breast, lung, colon, liver and kidney. The 26 cancer cell lines were derived from melanoma and cancers of the brain, breast, lung, colon, pancreas and prostate. All 5 MMR genes were ubiquitously expressed in all normal cell lines tested, suggesting their housekeeping roles. Aberrant MMR gene expression was only observed in the colon cancer cell lines. Two previously uncharacterized colon cancer cell lines did not express hMLH1. These data suggest that this nonradioactive multiplex RT-PCR assay for MMR gene expression may be useful for fast screening for genetic alterations that may affect gene expression and so may aid molecular analysis of MMR-related colon cancer.

Adaptor Proteins, Signal Transducing↗
PubMed1992

Goniodysgenesis of the eye with arthrogryposis multiplex congenita.

We report an autopsy case of 4-day-old male infant with arthrogryposis multiplex congenita studied by histological methods in respect to the ocular tissues. The eye examined was normal in size, and the retina, optic nerve, choroid, sclera and extracocular muscle showed no remarkable histological changes. However, the cornea was thickened, and the iris, trabecular tissue and ciliary body showed maldevelopmental changes, which were all compatible with goniodysgenesis. The clinical data of this case did not demonstrate any signs of congenital glaucoma, but the present study indicates that an infant with arthrogryposis multiplex congenita has a potential for infantile glaucoma with goniodysgenesis.

Abnormalities, Multiple↗
PubMed2005

Multiplex bead immunoassay analysis of aqueous humor reveals distinct cytokine profiles in uveitis.

PURPOSE: To extensively characterize the complex network of cytokines present in uveitis aqueous humor (AqH), and the relationships between cytokines and the cellular infiltrate. METHODS: AqH from noninflammatory control subjects and patients with idiopathic, Fuchs' heterochromic cyclitis (FHC), and herpes-viral or Behçet's uveitis were analyzed for IL-1beta, -2, -4, -5, -7, -8, -10, -12, -13, -15, TNFalpha, IFNgamma, CCL2 (MCP-1), CCL5 (RANTES), CCL11 (Eotaxin), TGFbeta2, and CXCL12 (SDF-1), using multiplex bead immunoassays. The cellular infiltrate was also determined for each sample. RESULTS: Idiopathic uveitis AqH, compared with noninflammatory controls, was characterized by high levels of IL-6, IL-8, CCL2 and IFNgamma, the levels of which correlated with each other. For IL-6 and IL-8 these levels were proportional to the number of neutrophils present. By contrast, the levels of both TGFbeta2 and CXCL12 decreased in idiopathic uveitis AqH with increasing inflammation. Cluster analysis showed a degree of segregation between noninflammatory and idiopathic uveitis AqH. Further examination using random forest analysis yielded a complete distinction between these two groups. The minimum cytokines required for this classification were IL-6, IL-8, CCL2, IL-13, TNFalpha, and IL-2. CONCLUSIONS: Application of multiplex bead immunoassays has allowed us to identify distinct patterns of cytokines that relate to both clinical disease and the cellular infiltrates present. Bioinformatics analysis allowed identification of cytokines that differentiate idiopathic uveitis from noninflammatory control AqH and are likely to be important for the pathogenesis of uveitis.

Adolescent↗
PubMed2001

Acetylcholine receptor delta subunit mutations underlie a fast-channel myasthenic syndrome and arthrogryposis multiplex congenita.

Limitation of movement during fetal development may lead to multiple joint contractures in the neonate, termed arthrogryposis multiplex congenita. Neuromuscular disorders are among the many different causes of reduced fetal movement. Many congenital myasthenic syndromes (CMSs) are due to mutations of the adult-specific epsilon subunit of the acetylcholine receptor (AChR), and, thus, functional deficits do not arise until late in gestation. However, an earlier effect on the fetus might be predicted with some defects of other AChR subunits. We studied a child who presented at birth with joint contractures and was subsequently found to have a CMS. Mutational screening revealed heteroallelic mutation within the AChR delta subunit gene, delta 756ins2 and delta E59K. Expression studies demonstrate that delta 756ins2 is a null mutation. By contrast, both fetal and adult AChR containing delta E59K have shorter than normal channel activations that predict fast decay of endplate currents. Thus, delta E59K causes dysfunction of fetal as well as the adult AChR and would explain the presence of joint contractures on the basis of reduced fetal movement. This is the first report of the association of AChR gene mutations with arthrogryposis multiplex congenita. It is probable that mutations that severely disrupt function of fetal AChR will underlie additional cases.

Action Potentials↗
PubMed1997

A two-dimensional array for B-mode and volumetric imaging with multiplexed electrostrictive elements.

A 2:1 multiplexed 2-D array has been developed that has a sparse element pattern designed for real time volumetric imaging and an alternate element pattern designed for B-mode imaging. For volumetric imaging, a small aperture was used to provide a wide transmit beam, allowing multiple beams to be received simultaneously. For B-mode imaging, a larger aperture with a more narrow transmit beam was used to improve image quality. Sparse random element patterns were evaluated by beamplot and cyst image simulations. Using the alternate element pattern for B-mode imaging, simulated cyst contrast was improved by 28%. The multiplexed transducer was fabricated using an electrostrictive material in which array elements were activated and deactivated by a dc bias field. The transducer had a 3.4 MHz center frequency with 46% bandwidth, which was consistent with KLM simulations. The high dielectric constant of the electrostrictive material resulted in an element clamped capacitance of 14.3 pF versus 2 pF for a convention PZT element. An output off isolation of -35 dB was measured for transmit and -61 dB for receive. The array was integrated with the volumetric scanner and used to make real time images of a cyst phantom. The images showed improved cyst contrast using the alternate aperture for B-mode imaging.

Image Processing, Computer-Assisted↗
PubMed2005

Neurogenic arthrogryposis multiplex congenita and velopharyngeal incompetence associated with chromosome 22q11.2 deletion.

We report a case of neurogenic arthrogryposis multiplex congenita and velopharyngeal incompetence in association with a chromosome 22q11.2 deletion in a 5-month-old boy, the only child of a non-consanguineous couple without relevant antecedents. Specifically, polymerase chain reaction amplification of microsatellite markers revealed a noninherited microdeletion in position D22S306. This phenotype has not been reported previously in association with chromosome 22q11.2 deletions, and these findings raise the possibility that at least some cases of neurogenic arthrogryposis multiplex congenita might be due to genetic defects of this type.

Arthrogryposis↗
PubMed1996

Mononeuritis multiplex in systemic lupus erythematosus: response to pulse intravenous cyclophosphamide.

Severe mononeuritis multiplex in systemic lupus erythematosus (SLE) is quite rare and almost always accompanied by evidence of active disease in other organs, although occasionally it may be the presenting feature of the disease. We describe here two patients with SLE who presented a severe mononeuritis multiplex secondary to a necrotizing vasculitis which respond successfully to therapy with intravenous cyclophosphamide, and review the literature of similar cases.

Adult↗
PubMed2001

Multiplex polymerase chain reaction assay for identification of enterotoxigenic Escherichia coli strains.

A multiplex polymerase chain reaction (PCR) system was developed for identification of enterotoxigenic Escherichia coli (ETEC) strains and to differentiate them from other gram negative enteric bacteria. This test simultaneously amplifies heat-labile (LTI) and heat-stable (STI and STII) toxin sequences and the E. coli-specific universal stress protein (uspA). The specificity of the method was validated by single PCR tests performed with the reference E. coli and non-E. coli strains and with bacteria isolated from pig feces. The multiplex PCR allowed the rapid and specific identification of enterotoxin-positive E. coli and may be used as a method for direct determination of ETEC and to differentiate them from other E. coli and gram-negative enteric isolates.

Animals↗
PubMed2002

Detection and differentiation of North American and European genotypes of porcine reproductive and respiratory syndrome virus in formalin-fixed, paraffin-embedded tissues by multiplex reverse transcription-nested polymerase chain reaction.

North American and European genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) were distinguished in formalin-fixed, paraffin-embedded tissues from PRRSV-infected pigs by multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR). A method based on xylene deparaffinization followed by proteinase K digestion yielded RNA of quality for reliable and consistent RT-nPCR analyses. The PRRSV nucleic acid was detected in lung, mediastinal lymph node, tonsil, and liver samples from pigs inoculated with North American strain virus, European strain virus, or both North American and European strains. All 30 archival formalin-fixed, paraffin-embedded lung tissues from pigs naturally infected with PRRSV were positive for PRRSV by RT-nPCR amplication and gave a pattern that corresponded to the North American genotype. Multiplex RT-nPCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for the detection and differentiation between North American and European genotypes of PRRSV.

Animals↗
PubMed2005

Use of a multiplex polymerase chain reaction to rapidly differentiate Neospora caninum from Toxoplasma gondii in an adult dog with necrotizing myocarditis and myocardial infarct.

This report describes a 3-year-old male castrated Mastiff dog that died unexpectedly with locally extensive, acute, necrotizing myocarditis and myocardial infarction. Intralesional protozoal tachyzoites in the affected myocardium were confirmed to be Neospora caninum by a novel multiplex polymerase chain reaction (PCR) and immunohistochemistry. Protozoal organisms were not identified in other tissues by histology, immunohistochemistry, or PCR. The multiplex PCR assay was used to quickly provide preliminary results on fresh myocardium to differentiate N. caninum and Toxoplasma gondii. Neosporosis is an uncommon cause of myocarditis in adult dogs and differential diagnoses for myocarditis in this population of dogs are reviewed.

Animals↗