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PubMed2004

A multiplex microsphere bead assay for comparative RNA expression analysis using flow cytometry.

Comparative gene-expression profiling is an important tool in understanding molecular signatures of complex diseases as well as the responses of cells and tissues to external factors. With increasing microarray data, disease-specific molecular patterns are emerging but the acquisition of these data is expensive, difficult to customize and not well standardized. Once genome-wide scans identify differentially expressed genes in a given disease, cheaper, more easily customized methods will be needed for evaluating the expression of these genes in large population samples. Here we describe a novel multiplex microsphere bead assay (MBA) to compare gene expression levels. To test this assay we evaluated the expression levels of four transcripts (BRCA1, MGB1, DLG1 and ACT1) in normal and cancerous mammary tissue. The results were consistent with those generated by quantitative real-time PCR.

Actins↗
PubMed2005

High-throughput and multiplexed protein array technology: protein-DNA and protein-protein interactions.

Miniaturized protein arrays address protein interactions with various types of molecules in a high-throughput and multiplexed fashion. This review focuses on achievements in the analysis of protein-DNA and protein-protein interactions. The technological feasibility of protein arrays depends on the different factors that enable the arrayed proteins to recognize molecular partners and on the specificity of the interactions involved. Proteome-scale studies of molecular interactions require high-throughput approaches for both the production and purification of functionally active proteins. Various solutions have been proposed to avoid non-specific protein interactions on array supports and to monitor low-abundance molecules. The data accumulated indicate that this emerging technology is perfectly suited to resolve networks of protein interactions involved in complex physiological and pathological phenomena in different organisms and to develop sensitive tools for biomedical applications.

Cell-Free System↗
PubMed2006

FOXE1 gene mutation screening by multiplex PCR/DHPLC in CHARGE syndrome and syndromic and non-syndromic cleft palate.

Denaturing high-performance liquid chromatography (DHPLC) has established itself as one of the most powerful tools for DNA variation screening. FOXE1, a highly GC-rich gene involved in syndromic cleft palate, is under investigation in thyroid dysgenesis, nonsyndromic cleft palate and squamous cell carcinoma. A technique for fast and simultaneous detection of sequence variants in the entire coding region of the FOXEl gene based on multiplex PCR/DHPLC is presented here. Given its characteristics of high sensitivity and rapidity, the testing strategy developed by us appears to be a reliable approach for FOXE1 analysis in the screening of a large population at risk.

Base Sequence↗
PubMed2004

Evaluation of a multiplex reverse transcriptase PCR ELISA for the detection of nine respiratory tract pathogens.

BACKGROUND: A multiplex reverse transcription (RT) polymerase chain reaction combined with a microwell hybridization assay (m-RT-PCR-ELISA) was previously developed to detect nine different microorganisms: enterovirus (EV), influenza virus type A (IVA) and type B (IVB), respiratory syncytial virus (RSV), parainfluenzavirus type 1 (PIV1) and type 3 (PIV3), adenovirus (AV), Mycoplasma pneumoniae (Mpn), Chlamydia pneumoniae (Cpn) in a single test. These organisms do not usually colonize the respiratory tract of humans, but, if present, it may be assumed they are involved in respiratory disease. OBJECTIVES AND STUDY DESIGN: The m-RT-PCR-ELISA was tested on (i) culture supernatants of unknown contents, (ii) by determining the analytical sensitivity of 10-fold serial dilutions of culture supernatants and (iii) by determining clinical sensitivity in a retrospective study on 411 clinical specimens. The specimens were re-tested in parallel by m-RT-PCR-ELISA versus the gold standard culture and immunfluorescence, and versus individual RT-PCR. RESULTS: (i) The 9-valent m-RT-PCR-ELISA shows 83% to 100% concordant results on 103 culture supernatants containing different organisms. (ii) The analytical sensitivity was as follows: higher sensitivity of the 9-valent m-RT-PCR-ELISA in comparison to culture in the cases of PIV3, IVA and IVB (factor 10) and AV and EV (factor 100), and lower sensitivity in case of RSV and PIV1 (factor 10). (iii) The agreement with the gold standard in the kappa statistic was excellent for RSV (kappa = 0.937), IVA (kappa = 0.940), very good for PIV1 (kappa = 0.914), IVB (kappa = 0.907) and satisfactory for PIV3 (kappa = 0.410). For AV, EV and Mpn the m-RT-PCR-ELISA preliminary could be qualified as very good, based on the data derived on culture supernatants. Information about the validity for Cpn is limited. CONCLUSION: The m-RT-PCR-ELISA is a feasible, sensitive and specific method for detection of a broad spectrum of organisms. It is suitable for individual as well as epidemiological diagnosis.

Adenoviridae↗
PubMed2004

Multiplex nested PCR (MNP) assay for the detection of 15 high risk genotypes of human papillomavirus.

BACKGROUND: Human papillomavirus (HPV) is now recognized as the causative agent in cervical cancer. The HPV genotypes that infect the genital region have been classified into high and low risk types according to their oncogenic potential. There is still uncertainty regarding rare HPV genotypes, however the types considered high risk in this study are: HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and 70. OBJECTIVES: We have set out to develop a multiplex nested PCR (MNP) assay with primers directed at the early region of the HPV genome to detect 15 high risk HPV (HRHPV) genotypes. Since it is known that the late region of HPV is lost on integration into the host cell genome, the primers are directed at the early region of the HPV genome so as to ensure the detection of integrated virus, in the absence of the episomal form of the virus. STUDY DESIGN: Primers were designed to detect specifically the high risk HPV in the MNP assay. The MNP assay was compared to a generic mucosal HPV nested PCR and another nested HRHPV PCR assay. DNA sequencing was carried out on the samples tested and matched with the PCR results. RESULTS: The MNP assay demonstrated that it was able to detect all 15 HRHPV types and was positive for more CIN1, CIN2 and CIN3 cases than the other nested HRHPV PCR. Further to this, the PCR product sizes differ for most of the HRHPV types detected in this system, so it is possible to type most of these HRHPV by the molecular size of the PCR products. CONCLUSION: The MNP assay detects 15 currently recognized HRHPV and could be very useful, in conjunction with the Pap smear, as a screening assay or to help manage Pap smears of uncertain cytology.

Australia↗
PubMed2005

Multiplex real time RT-PCR for the detection and quantitation of norovirus genogroups I and II in patients with acute gastroenteritis.

BACKGROUND: Conventional reverse transcription-polymerase chain reaction (Con RT-PCR) assay to detect norovirus is a complex multi-step procedure that requires gel electrophoresis as well as hybridization or sequencing to confirm the results. OBJECTIVE: To develop and evaluate a multiplex real time RT-PCR (Mrt RT-PCR) assay that detect and quantify norovirus GI and GII with a single amplification and detection step. STUDY DESIGN: The primers and TaqMan probes for the Mrt RT-PCR were selected from the ORF1-ORF2 junction region. A total of 97 stools from 41 gastroenteritis outbreaks and 726 stools from children with sporadic diarrhoea were used for this study. RESULTS: For the 97 outbreak samples, norovirus were detected in 61 of the 69 previously tested positive and 11 of the 28 previously tested negative samples. Eight samples that tested positive for GII by Con RT-PCR but negative by the Mrt RT-PCR also tested negative by a Light Cycler RT-PCR assay. Eighty-two GII and two GI were detected in the 726 sporadic samples. Random primers were more sensitive than specific primers in the cDNA synthesis. The two-step assay using the random primers in RT reaction was 100 times more sensitive than the one-step assay. The Mrt RT-PCR had the same sensitivity as that using two real time RT-PCR for separate detection of GI and GII. A wide dynamic range was obtained with the two-step assay, detecting from 3000 to 3x10(11) of copies RNA/g stool. Very good precision was observed with no cross-reaction with other enteric viruses. The new assay is able to detect both GI and GII in one reaction and brings a cost reduction of approximately 40% compared to separate reactions for GI and GII. CONCLUSIONS: The assay has good precision, sensitivity and specificity and is cost-effective as a routine diagnostic test.

Caliciviridae Infections↗
PubMed2006

Multiplex-PCR and oligonucleotide microarray for detection of eight different herpesviruses from clinical specimens.

BACKGROUND: Human herpesviruses cause clinically important diseases, e.g. infections of the central nervous system. New diagnostic tools are required for rapid and reliable detection of these viruses. OBJECTIVES: A microarray-based method was designed for detection of eight human herpesviruses in cerebrospinal fluid (CSF), whole blood, plasma, serum and proficiency-testing specimens. STUDY DESIGN: Herpes simplex type 1 and 2, varicella-zoster, cytomegalo-, Epstein-Barr and human herpes viruses 6A, 6B and 7 were amplified from clinical specimens by two multiplex-PCRs and transcribed to single-stranded RNAs which were hybridized to oligonucleotides on microarray. The results were compared to those from conventional PCR. In total, 227 specimens were tested including 23 CSF, 10 whole blood, 73 plasma, 10 proficiency-testing samples and 111 negative control samples. RESULTS: Concordant results were obtained in 214/227 (94%). Microarray detected 10 possible double and one triple infection. Negative control samples (70 serum, 30 CSF and 11 proficiency-testing samples) were all negative. CONCLUSIONS: Microarray is suitable for detection of multiple herpesviruses in clinical samples.

Herpesviridae↗
PubMed2006

Rapid detection of Norovirus based on an automated extraction protocol and a real-time multiplexed single-step RT-PCR.

BACKGROUND: Molecular diagnosis of Norovirus infection can be a complex multistep process, which requires significant user intervention and expertise, and is not amenable to automation without extensive validation and optimization. OBJECTIVES: To develop a real-time multiplexed RT-PCR assay with automated sample preparation that requires only a single-step and a single-tube for reverse transcription, amplification, and detection while exceeding the sensitivity of conventional PCR for broad-spectrum Norovirus detection. STUDY DESIGN: Limit of detection was assessed against dilutions of clinical specimens. Fifty archived extractions were used to compare TaqMan sensitivity with either a separate RT using random primers or a single-step RT-PCR. The sensitivity of the novel assay was compared with conventional RT-PCR using 100 specimens from gastroenteritis cases. RESULTS: Automated extraction reduced RNA recovery by 0.5 logs compared to manual extraction but was more effective at removing PCR inhibitors from stool specimens. The optimized single-step real-time RT-PCR demonstrated no reduction in sensitivity. Together, the sensitivity of the novel assay was 19% higher than manual extraction with conventional RT-PCR. CONCLUSIONS: A semi-automated and simplified molecular diagnostic protocol for the rapid detection of Norovirus has been achieved. PCR inhibitors are present in human fecal specimens and cause a significant problem for Norovirus detection by RT-PCR.

Caliciviridae Infections↗
PubMed2005

Genome profiling of melanocytic tumors using multiplex ligation-dependent probe amplification (MLPA): Its usefulness as an adjunctive diagnostic tool for melanocytic tumors.

BACKGROUND: The histopathology of melanocytic tumors sometimes presents diagnostic problems. Applicable parameters other than routine pathology are needed. OBJECTIVE: We assessed the feasibility of multiplex ligation-dependent probe amplification (MLPA), a novel PCR-based genome profiling method, in the classification of melanocytic tumors. METHOD: We extracted DNA from paraffin-embedded tissue sections of 24 primary melanomas, 14 Spitz nevi and 17 common melanocytic nevi. We analyzed the copy number gains or losses of a total of 76 genes spanning almost all chromosome arms using commercially available MLPA kits. RESULTS: Although four melanocytic nevi and three Spitz nevi did not yield sufficient DNA for reliable analysis due to small tumor size, the MLPA analysis was feasible and applicable to the remaining 88% of samples. We found multiple genetic aberrations in primary melanomas. The total number of aberrations in each tumor ranged from 1 to 32 (average, 12.04). All but two melanomas showed aberrations at more than three genetic loci. Seventeen (70.8%) of the 24 melanomas showed a copy number loss of either the CDKN2A or CDKN2B gene on chromosome 9p21. All the Spitz nevi and 7 (50%) of 14 common melanocytic nevi had copy number changes at one or two gene loci (average, 1.04). The receiver operator characteristic curve analysis showed that the threshold value of copy number aberrations corresponding to 98% specificity for melanoma was 2.42 and the sensitivity using this threshold value was 92.5%. CONCLUSIONS: MLPA could be used as an adjunctive diagnostic tool for melanocytic tumors.

Cell Cycle Proteins↗
PubMed2004

Arthrogryposis multiplex congenita: functional classification and the AMC disc-o-gram.

The arthrogryposis multiplex congenita (AMC) disc-o-gram is presented as a way to measure the effect of hand therapy, splinting and/or surgery on the AMC patient. It also allows comparative measurement of passive motion (in the baby), active motion (in the young child) and function (in the older child and adult). These measurements are related to a new classification system which, as movement and function improves, indicates the efficacy of management.

Adolescent↗
PubMed2005

Arthrogryposis multiplex congenita.

Since much confusion exists regarding arthrogryposis multiplex congenita (AMC), the President of the IFSSH commissioned the AMC Committee to compile a report on the various aspects of this condition. This report discusses all the facets of AMC, including definition, terminology, dermographics, aetiology, classification, clinical features, management, prognosis and further studies. Manipulation of the deformities starting soon after birth improves the range of motion, which, if surgery needs to be done, makes the operation less extensive. Sometimes surgery may not even be necessary. Early one-stage corrective surgery between 3 and 12 months of age is encouraged. This results in improved function and near normal cosmetic appearance. Late, piece-meal surgery results in disappointing outcome.

Arthrogryposis↗
PubMed2004

Multiplexed sandwich assays in microarray format.

Antibody arrays have evolved into powerful tools for quantifying proteins and qualifying their state of activation in complex biological samples. This level of analysis holds tremendous promise as part of a diagnostic or prognostic platform. In particular, multiplex sandwich ELISA assays performed with microarrays in the wells of multi-well plates enable high-throughput analysis of multiple samples with multivariate readouts. Here, we compare and review recent advances in antibody microarray technology and describe its promises towards the systematic analysis of complex biological samples, ranging from profiling patient sera to studying intracellular signaling.

Animals↗
PubMed2004

Cost-effective genotyping of human MBL2 gene mutations using multiplex PCR.

Mannose-binding lectin (MBL) deficiency is associated with increased susceptibility to various infections and autoimmune disorders. It is caused by certain polymorphisms in the MBL2 gene promoter and mutations in the coding region of the gene. In this report, we present a novel, rapid, efficient and cost-effective method of two multiplex polymerase chain reactions (PCRs) for the assessment of three structural point mutations within exon 1 at codons 52, 54 and 57. Three additional PCR reactions for the detection of promoter polymorphisms at positions -550 and -221 were performed. MBL2 haplotypes in 359 individuals of the general Czech population were detected using this approach. The rare LYD haplotype was found in 1.1% of all alleles.

Amino Acid Sequence↗
PubMed2005

A multiplexed and miniaturized serological tuberculosis assay identifies antigens that discriminate maximally between TB and non-TB sera.

We have developed a multiplexed and miniaturized TB serological assay with the aim of identifying (combinations of) antigens that maximally discriminate between TB and non-TB patients. It features a microarray accommodating 54 TB antigens, less than 1 microl serum consumption and an indirect immunofluorescence detection protocol. With a panel of 20 TB and 80 non-TB sera we ranked combinations of TB antigens with respect to sensitivity and specificity of TB detection by means of logistic step-forward regression analysis. The highest-ranking TB antigen combination had an area-under-the-curve of the receiver-operator-characteristics (ROC) of 0.95. We also identified an antigen that on its own provided good specificity and sensitivity of TB detection (Ara6-BSA; area-under-the-ROC curve: 0.90). These area-under-the-ROC curve values are exceptionally high for a serological TB assay. We conclude that TB antigen microarrays permit rapid identification of TB antigens that, either alone or in combination, discriminate maximally between TB and non-TB patients and that such identification provides an excellent starting point for developing point-of-care diagnostic assays.

Antibodies, Bacterial↗
PubMed2004

Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue.

The ability to simultaneously quantify multiple signaling molecule protein levels from microscopic neural tissue samples would be of great benefit to deciphering how they affect brain function. This follows from evidence that indicates signaling molecules can be pleiotropic and can have complex interactive behavior that is regionally and cellularly heterogeneous. Multiplexed examination of tissue proteins has been exceedingly difficult because of the absence of available techniques. This void now has been removed by the commercial availability of bead-based immunoassays for targeted proteins that allow analyses of up to 100 (6-150 kDa) proteins from as little as 12 microl. Thus far used only for sera (human and mouse) and culture media, we demonstrate here that sensitive (as low as 2 pg/ml), wide-ranging (up to 2-32 000 pg/ml), accurate (8% intra-assay covariance) and reliable (4-7% inter-assay covariance) measurements can be made of nine exemplary cytokines (e.g., IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma, TNF-alpha) simultaneously not only from rat serum but, for the first time, also brain tissue. Furthermore, we describe animal handling procedures that minimize stress as determined by serum glucocorticoid levels since they can influence cytokine expression.

Animals↗
PubMed2005

Multiplexed cytokine detection in microliter microdialysis samples obtained from activated cultured macrophages.

Microdialysis sampling probes were used to collect cytokine samples from lipopolysaccharide (LPS)-stimulated macrophages. The probes were immersed into cell culture wells containing either RAW 264.7 or isolated peritoneal macrophages. Dialysates (15 microL) from these wells were subjected to a multiplexed cytokine sandwich immunoassay platform analyzed by flow cytometry that measures up to six separate cytokines, interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), interferon-gamma (IFN-gamma), macrophage chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-alpha) in a single 15-muL sample. In vitro microdialysis sampling relative recovery experiments showed that only IFN-gamma, IL-6, MCP-1, and TNF-alpha could be recovered across a commercially-available 100-kDa MWCO microdialysis membrane. Eleven hours after LPS addition (1 microg/mL), RAW 264.7 macrophages secreted greater than 8000 pg/mL of TNF-alpha and greater than 1000 pg/mL MCP-1. With the peritoneal macrophages, greater than 6000 pg/mL of IL-6, MCP-1, and TNF-alpha were obtained. The maximum dialysate concentrations obtained from the RAW macrophages were 1300 pg/mL for TNF-alpha and 55 pg/mL for MCP-1. Maximum cytokine concentrations from peritoneal macrophage dialysates reached approximately 2000 pg/mL, 1100 pg/mL and 500 pg/mL for TNF-alpha, MCP-1 and IL-6, respectively. Microdialysis sampling allowed for 20-min samples to be collected during the cytokine release from the activated macrophages. These results demonstrate that microdialysis sampling can be used for collection of selected cytokines with improved temporal resolution.

Animals↗
PubMed2005

Validation and comparison of luminex multiplex cytokine analysis kits with ELISA: determinations of a panel of nine cytokines in clinical sample culture supernatants.

PROBLEM: Analyses of the expression pattern of multiple cytokines are frequently required for characterization of the status of the immune system as it pertains to Th type bias and intrinsic levels of inflammation. Classically, analysis of cytokine expression patterns has been performed by enzyme-linked immunosorbent assays (ELISA) for each separate analyte. A new technology, Luminex MAP, facilitates the simultaneous evaluation of multiple immune mediators with advantages of higher throughput, smaller sample volume, and lower cost. Validation of this technology has been limited to small sample sets, limited use of clinical study specimens, and use of non-commercial reagents. METHODS: Ninety-six specimens from women over the course of their respective pregnancies were evaluated for cytokine concentrations using commercially available ELISA kits and commercially available Luminex MAP kits according to the manufacturers' directions. Correlations between data sets were evaluated using Pearson's correlation coefficient (r). RESULTS: Excellent correlations were demonstrated for IL-1 beta, IL-4, IL-5, IL-6, IL-10, IFN gamma, and TNF alpha, in contrast to IL-12 p 70 and IL-13. CONCLUSIONS: Luminex multiplex technology has distinct advantages and is a valid alternative method to ELISA for the evaluation of the majority of cytokines tested and for the characterization of immune system status.

Cytokines↗
PubMed2004

Simultaneous detection of bovine arboviruses using single-tube multiplex reverse transcription-polymerase chain reaction.

Single-tube multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay was developed to detect and identify arboviruses in infected cell-culture fluids and field specimens. The technique was equally sensitive for detecting five different viruses in cell cultures, namely the Chuzan, Ibaraki, and Bluetongue viruses belonging to Orbivirus, and the Akabane virus and Peaton virus belonging to Orthobunyavirus, and was less sensitive than former viruses for detecting Aino virus belonging to Orthobunyavirus. The mRT-PCR reliably detected 0.6-10(3.1) median tissue culture infective doses. The mRT-PCR readily identified viruses by discriminating the size of their amplified gene products. The technique was as sensitive as virus isolation in detecting single infected plasma in five plasmas from sentinel cattle and in detecting two infectious homogenates in eight homogenates of Culicoides biting midges. The mRT-PCR may be a sensitive and rapid assay for surveillance of bovine arboviruses in field specimens.

Animals↗