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At least 811 records · Page 45Linked to original sources
PubMed2005

Development of a multiplex nested consensus PCR for detection and identification of major human herpesviruses in CNS infections.

BACKGROUND: Rapid, sensitive and economical detection and identification of human herpesviruses as causative agents of central nervous system (CNS) infections are of clinical importance. The traditional methods for the detection of herpesviruses in CNS infections all suffer from limitations. PCR has a potential to overcome each of them. OBJECTIVES: The aims of this study were reducing the number of primers in multiplex PCR and increasing the sensitivity of the assay by nested PCR. STUDY DESIGN: A multiplex nested consensus PCR (MNC-PCR) was developed for the simultaneous detection of major human herpesviruses. A pair of conserved primers was designed for detection of HSV-1, HSV-2, CMV and EBV and another pair of conserved primers for nested PCR. For VZV, a different pair of primers was designed and another pair of primers for nested PCR. A reduction in the number of designed primer pairs (from five pairs to two in both stages of PCR) is an advantage in this assay. One hundred forty-seven cerebral spinal fluid (CSF) samples from patients that showed clinical manifestation of CNS infections were tested. Results of MNC-PCR in CSF samples were compared with those of single PCR assay for each individual DNA virus. Sensitivity of the assay was determined with a plasmid containing VZV DNA binding protein gene and another plasmid for HSV-1 DNA polymerase gene. False negative results (due to the presence of inhibitor of DNA amplification in CSF samples) were avoided by the inclusion of beta2-microglobulin primers in the MNC-PCR assay as an internal control. RESULTS: Positive results were obtained in 20 CSF samples (8 HSV-1, 2 HSV-2, 4 CMV, 3 VZV, 3 HSV-1/CMV, CMV/VZV and HSV-1/EBV coinfections). The comparison between single PCR and MNC-PCR showed a marked increase in sensitivity of MNC-PCR test, since six negative samples in single PCR proved positive in MNC-PCR (P<0.005). Sensitivity was determined 1-5 plasmid copies for VZV and 50-100 plasmid copies for HSV-1. CONCLUSIONS: The MNC-PCR assay presented in this study can provide a rapid, sensitive and economical method for detection of viral infections and is applicable to small volumes of CSF samples.

Adult↗
PubMed2004

Analysis of the HIV-1 gp41 specific immune response using a multiplexed antibody detection assay.

A fluorescence-based, multiplexed, antibody-binding and mapping assay was developed to characterize antibody responses in HIV-1-infected individuals to the ectodomain of the HIV-1 gp41 envelope glycoprotein. The antigen panel included intact recombinant gp41, the fusion peptide region, the polar region, the N-heptad region, the C-heptad region as well as overlapping epitopes in the 2F5 and 4E10 monoclonal antibody-binding regions. The panel included both native and constrained peptides specifically designed to mimic putative gp41 prefusion and fusion intermediates. The results of these analyses revealed a broad pattern of immune responses against the test antigens, suggesting that none of these gp41 regions are immunologically silent. The HIV-1-positive sera were also evaluated using infectivity inhibition assays. No correlation was evident between the breadth or magnitude of specific anti-gp41 reactivities and virus neutralization potency. These evaluations demonstrated the substantial potential of the multiplexed antibody binding and mapping assay for rapid and sensitive analysis of complex antibody responses.

Amino Acid Sequence↗
PubMed2004

A novel method of Multiplexed Competitive Antibody Binning for the characterization of monoclonal antibodies.

We have developed a novel method of high-throughput Multiplexed Competitive Antibody Binning (MCAB). Using only a small amount of antibody and antigen, this method enables the sorting of a large, complex panel of monoclonal antibodies into different bins based on cross-competition for antigen binding. The MCAB assay builds on Luminex multiplexing bead-based technology to detect antibody competition. Because of its high sensitivity, the MCAB method is immediately applicable after identification of antigen-positive mAbs, providing information useful for advancing mAb candidates into further testing. The MCAB assay also can be used for sorting mAbs into binding groups after screening for functional activity.

Animals↗
PubMed2005

Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies.

Cytokines, chemokines and soluble adhesion molecules interact in a complex network within the immune system. Fingerprinting of these proteins may allow the use of these proteins as biomarkers for identification of disease, disease subtyping and monitoring therapeutic interventions. We developed a multiplex immunoassay (MIA) for the detection of 30 proteins in a variety of human body fluids such as plasma and synovial fluid (SF). The measurement of these proteins is hampered by the presence of human (auto-) antibodies, which can cause non-specific binding. We have validated a novel approach for the removal of interfering immunoglobulins using pre-absorption with protein-L. Interfering (auto-) antibodies, such as rheumatoid factor (RF), were removed using three methods; polyethylene glycol (PEG) precipitation, pre-absorption with human gamma-globulin or pre-absorption with protein-L. A significant decrease of RF was observed after a 2 h incubation with protein-L. RF IgM levels were reduced by 89% whereas total IgM, IgG and IgA levels were reduced by 60%. Residual immunoglobulins were blocked with rodent serum and did not interfere with the multiplex immunoassay. Comparing the MIA with a conventional enzyme-linked immunosorbent assay (ELISA) using a panel of spiked plasma samples resulted in correlation coefficients for all mediators between R2 = 0.88 and R2 = 0.99. Intra-assay variance was less than 10% whereas inter-assay variance ranged between 6% and 16%. Pathological samples with heterophilic antibodies hamper immunoassays such as ELISA and MIA. We show that pre-absorption with protein-L is a powerful tool for removal of interfering immunoglobulins from human bodily fluids to be used in immunoassays for studying changes in protein patterns.

Antibodies, Heterophile↗
PubMed2006

Multiplexed PrEST immunization for high-throughput affinity proteomics.

Monospecific antibodies dfdfdfdf (msAbs) generated through antigen specific purification of polyclonal antisera are valuable tools in proteome analyses. However, proteome wide generation of msAbs would require extensive immunization programs. Therefore, it would be desirable to develop efficient immunization and purification methods to reduce the number of animals needed for such antibody-based research. Here we describe a multiplex immunization strategy for generation of msAbs towards recombinantly produced human protein fragments, denoted PrESTs. Antisera from rabbits immunized with a mixture of two, three, five and up to ten different PrESTs have been purified by a two-step immunoaffinity-based protocol and the efficiency of the purification method was analyzed using a two-color protein array concept. The obtained results showed that almost 80% of the animals immunized with antigens composed of two or three different PrESTs yielded antibodies recognizing all the included PrESTs. Furthermore, the modified two-step purification method effectively eliminated all background binding and produced pure antibody pools against individual PrESTs. This indicates that the multiplexed PrEST immunization strategy described here could become useful for high-throughput antibody-based proteomics initiatives, thus significantly reducing the number of animals needed in addition to providing a more cost-efficient method for production of msAbs.

Animals↗
PubMed2006

Development of a multiplexed fluorescent immunoassay for the quantitation of antibody responses to group A streptococci.

The host immunologic response to group A streptococcal infections gives rise to numerous antibodies directed against cellular and extracellular bacterial antigens. For determining individual immune status, or studying the pathogenesis of group A streptococcal associated diseases, such as acute rheumatic fever (ARF), an assay capable of determining antibodies responses to multiple antigens would be of great advantage. We have developed a microsphere based, multiplexed immunoassay for the simultaneous quantitation of antibodies to nine different extracellular, ARF related tissue and group A streptococci specific antigens using only 5 microl of sample. Through the selection of microspheres and serum diluent, non-specific antibody binding was reduced by 17%. Different formulations of the coupling buffer were found to greatly influence the efficiency of coupling antigens to the carboxylated microspheres. Monoclonal antibodies against the different antigens demonstrated assay specificity as well as sensitivities of less than 1 ng/ml of antibody. This multiplexed assay should be a powerful research and clinical tool in determining antibody responses to group A streptococcal infections and in potentially determining the role of a variety of cross-reactive antigens in rheumatic fever and rheumatic heart disease.

Antibodies, Bacterial↗
PubMed2006

A multiplexed proteomics approach to differentiate neurite outgrowth patterns.

We report here a method for proteomics pattern discovery by utilizing a self-organizing map approach to analyze data obtained from a novel multiplex iTRAQ proteomics method. Through the application of this technique, we were able to delineate the early molecular events preceding dorsal root ganglia neurite outgrowth induced by either nerve growth factor (NGF) or an immunophilin ligand, JNJ460. Following pattern analysis we discovered that each neurotrophic agent promoted mostly distinct increases in protein expression with few overlapping patterns. In the NGF-treated group, proteins possessing "biosynthesis function" (p < 0.002) and "ribosome localization" (p < 0.0003) were increased, while proteins promoting "organogenesis" (p < 0.004) and related "signal transduction" (p < 0.008) functions were notably increased in the JNJ460-treated group. This study suggests that the properties of neurite outgrowth triggered by NGF and JNJ460 can be distinguished at the proteome level. Multiplexed proteomics analysis, along with pattern discovery bioinformatics tools, has the capability to differentiate subtle neuroproteomics patterns.

Animals↗
PubMed2006

Paraneoplastic mononeuritis multiplex in non-small-cell lung carcinoma.

A 60-year-old man developed two selective peripheral mononeuropathies of the peroneal and later the radial nerve, shortly after a diagnosis of large-cell lung carcinoma. Nerve conduction studies and electromyography confirmed isolated lesions in both nerves, and in the case of the peroneal nerve lesion, focal conduction block was localised to the level of the fibula neck. Subsequent magnetic resonance imaging of the lower limb excluded focal compression or malignant infiltration along the course of the peroneal nerve, and there was no signal change within the nerve, prompting a diagnosis of paraneoplastic mononeuritis multiplex. Anti-neuronal antibodies and serological markers of systemic vasculitis were negative. Neither the patient's large-cell lung carcinoma nor mononeuritis multiplex responded to chemotherapy, and he died within 6 months of the initial diagnosis.

Carcinoma, Non-Small-Cell Lung↗
PubMed2006

Multiplex measurement of cytokine/receptor gene polymorphisms and interaction between interleukin-10 (-1082) genotype and chorioamnionitis in extreme preterm delivery.

OBJECTIVES: To establish a multiplex amplification refractory mutation system (ARMS) in fluid and dried whole blood, and to perform a pilot study to examine the role for single-nucleotide polymorphisms (SNPs) of inflammation-associated genes (interleukin [IL]-1 and -10, tumor necrosis factor-alpha [TNFA], and toll-like receptor-4 [TLR4]) and their interaction with clinical chorioamnionitis (CAM) in prematurity. METHODS: We established a quadruplex ARMS to detect the four above SNPs. Fifty-four women delivered at gestational age less than 32 weeks and 83 healthy female volunteers were genotyped. We compared (1) mothers of preterm infants with volunteers, and (2) women delivered before 29 weeks' gestation (n = 29) with those delivered at 29 to 31 completed weeks (n = 25). RESULTS: Multiplex ARMS is feasible using both fluid and dried whole blood. We found no overall differences in genotype and allele frequencies between mothers of preterm infants and volunteers. Among women who had a preterm delivery, those with both CAM and IL10(-1082)*G allele, the risk for delivery before 29 weeks was markedly increased (odds ratio [OR] 22, 95% confidence interval [CI] 2.5 - 191). CONCLUSION: The presence of both CAM and IL10(-1082)*G might play a role in extreme preterm delivery less than 29 weeks.

Chorioamnionitis↗
PubMed2003

Detection of norovirus (GI, GII), Sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex PCR.

A reverse transcription (RT) single-round multiplex polymerase chain reaction (smPCR) assay was developed to detect simultaneously Norovirus genogroup I and II, Sapovirus and astrovirus. A total of 377 diarrhea stool samples (screened for rotavirus- and adenorivus-negative) from four regions in Japan during July 2000 to June 2001 were examined by RT-smPCR. The positive rate was 16.4% (62 out of 377 stool samples). Norovirus, Sapovirus and astrovirus were detected in 42, 16, 4 of 60 positive samples, respectively. Coinfection was not found in these samples. Infections occurred mainly in November, December and January. The key elements of the RT-smPCR are (i) the cDNA synthesis with the Superscript RTII and random primer at 42 degrees C for 1 h, at 99 degrees C for 5 min, and (ii) single-round multiplex PCR by using Taq polymerase mixed together with a mixture of four different primer pairs (G1-SKF/G1-SKR for Norovirus genogroup I, COG2F/G2-SKR for Norovirus genogroup II, SLV5317/SLV5749 for Sapovirus, PreCAP1/82b for astrovirus). All of the four primer pairs amplify the capsid region of target viral genome, produce four size-specific amplicons of 330, 387, 434, 719 bp for Norovirus genogroup I and II, Sapovirus and astrovirus, respectively. This assay provides a more rapid and efficient way to detect these viruses from fecal samples in a single test, and also offers the potential for their molecular detection in food and environmental samples.

Adolescent↗
PubMed2004

Detection and quantitation of akabane and aino viruses by multiplex real-time reverse-transcriptase PCR.

A multiplex, quantitive reverse-transcriptase real-time PCR, using MGB TaqMan chemistry, for detecting akabane virus (AKAV) and aino virus (AINV) is described. Each specific probe was labeled with a different fluorescent dye--VIC for detecting AKAV and 6-carboxy-fluorescein (FAM) for detecting AINV. All available sequences of viral S RNA were aligned and primers and probes were designed so that AKAV primers and probes would recognize all AKAVs but not AINV, and vice versa. The parameters for multiplex reactions enabled the detection of both viruses in one tube reaction with similar efficiency. To quantitate the viruses, cDNA amplicons containing the real-time amplicon were prepared using forward primers carrying the T7 promoter sequences. The cDNAs were used directly as templates for run-off transcription and 10-fold dilutions of the products served as standards to quantitate unknown viral samples. Using this system had shown that it could detect approximately 3-30 copies of viral S genome.

Animals↗
PubMed2004

A multiplex RT-PCR assay capable of distinguishing beet necrotic yellow vein virus types A and B.

A multiplex reverse-transcription polymerase chain assay (mRT-PCR) was developed, based on primers designed to distinguish the A and B types of beet necrotic yellow vein virus (BNYVV). RNA was extracted from 72 BNYVV isolates from Asia, Europe and North America, and the type of each isolate determined using an established detection method based on single strand conformation polymorphisms (SSCPs). An area of the 'triple gene block' region on RNA 2 was amplified and sequenced from 16 isolates of the A and B types. These sequences were aligned and two sets of PCR primers were designed to amplify unique areas common to each type. The A type assay produced a single 324 base-pair RT-PCR fragment when positive samples were amplified. The B type assay produced a 178 base-pair product from positive samples. No amplification was observed from healthy Chenopodium quinoa or sugar beet plants and from plants infected by others sugar beet soil-borne viruses. Fragment length differed sufficiently to allow both assays to be run in a single PCR tube. The results obtained using the new multiplex RT-PCR assay were consistent with those from the established SSCP method for all 72 reference samples.

Base Sequence↗
PubMed2005

Multiplex real-time PCR assay for simultaneous quantitation of human cytomegalovirus and herpesvirus-6 in polymorphonuclear and mononuclear cells of transplant recipients.

Human cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6) are two closely related viruses, which belong to the Herpesviridae family. Following primary infection, they are thought to persist for life as latent forms in mononuclear cells. HCMV and HHV-6 can cause considerable morbidity in immunocompromised individuals, such as transplant patients. A sensitive and specific LightCycler multiplex real-time PCR assay based on fluorescence energy transfer (known as FRET) was developed. This assay, by using two sets of hybridization probes specific for HHV-6 (A and B) and HCMV, can differentiate reliably and quantify simultaneously both viruses in order to diagnose reactivation processes. The assay was optimized and the lower limit of detection for both viruses was determined to be 10 viral genome copies per reaction. Both viruses were quantified in 83 peripheral blood mononuclear cells (PBMCs) and 87 polymorphonuclear leukocytes (PMNLs) collected from 32 transplant recipients. This multiplex real-time quantitative PCR was finally compared with two other quantitation and detection assays used daily in laboratory (PCR DIG detection and antigenemia for HCMV, TaqMan Assay for HHV-6). This technique can be useful for the differentiation and quantitation of HCMV and HHV-6 for monitoring transplant patients.

Adult↗
PubMed2006

A multiplex RT-PCR method for screening of reassortant live influenza vaccine virus strains.

A system based on reverse transcription polymerase chain reaction (RT-PCR) of the RNA genome was established to identify genetic composition of influenza viruses generated by reassortment between an attenuated donor virus and virulent wild type virus. The primers were designed, by multiple sequence alignment of variable regions, specific for cold-adapted donor virus HTCA-A 101, as compared to other influenza A viruses. The specificity of each primer set was confirmed and the primers were combined to perform RT-PCR in multiplex manner. The multiplex PCR was adopted to distinguish the 6:2 reassortant viruses containing six internal genome segments of attenuated donor virus and two surface antigens of virulent strain from the wild type viruses. The method allowed us to optimize the reassorting process on a routine basis and to confirm the selection of reassortant clones efficiently. The method is suitable for analyzing the contribution of specific gene segments for growth and attenuating characteristics and for generation of live attenuated vaccine by annual reassortment.

Adaptation, Physiological↗
PubMed2006

Improved detection of episomal Banana streak viruses by multiplex immunocapture PCR.

Banana streak viruses (BSV) are currently the main viral constraint to Musa germplasm movement, genetic improvement and mass propagation. Therefore, it is necessary to develop and implement BSV detection strategies that are both reliable and sensitive, such as PCR-based techniques. Unfortunately, BSV endogenous pararetrovirus sequences (BSV EPRVs) are present in the genome of Musa balbisiana. They interfere with PCR-based detection of episomal BSV in infected banana and plantain, such as immunocapture PCR. Therefore, a multiplex, immunocapture PCR (M-IC-PCR) was developed for the detection of BSV. Musa sequence tagged microsatellite site (STMS) primers were selected and used in combination with BSV species-specific primers in order to monitor possible contamination by Musa genomic DNA, using multiplex PCR. Furthermore, immunocapture conditions were optimized in order to prevent Musa DNA from interfering with episomal BSV DNA during the PCR step. This improved detection method successfully allowed the accurate, specific and sensitive detection of episomal DNA only from distinct BSV species. Its implementation should benefit PCR-based detection of viruses for which homologous sequences are present in the genome of their hosts, including transgenic plants expressing viral sequences.

Badnavirus↗
PubMed2004

A multiplex PCR assay for the identification of animal species in feedstuffs.

A multiplex Polymerase Chain Reaction (PCR) assay was applied to feedstuff analysis for the identification of the most used species in rendering plants (ruminant, poultry, fish and pork materials). Primers were designed in different regions of mitochondrial DNA (12S rRNA, tRNA Val and 16S rRNA) after alignment of the available sequences in the GenBank database. The primers generated specific fragments of 104-106, 183, 220-230 and 290 bp length for ruminants, poultry, fish and pork, respectively. The detection limit was 0.004% for fish primers and 0.002% for ruminants, poultry and pork primers. The multiplex PCR proposed in this study can be considered a valid alternative to the microscopic method for the detection of animal derived materials banned by a European Union Regulation as a preventive measure against the spread of Bovine Spongiform Encephalopathy.

Animal Feed↗
PubMed2004

A real-time multiplexed PCR assay for rapid detection and differentiation of Campylobacter jejuni and Campylobacter coli.

Campylobacter species are the leading agents of bacterial gastroenteritis worldwide. C. jejuni and C. coli together are responsible for more than 95% of all cases of Campylobacter-induced diarrheal disease in the United States. Detection of campylobacteria in clinical samples by conventional culture is problematic and slow due to their complex taxonomy, fastidious growth requirements, and biochemical inertness. The current study describes a rapid, sensitive, and specific real-time polymerase chain reaction (PCR) assay capable of detecting and differentiating C. jejuni (hippuricase gene, hipO) and C. coli (serine hydroxymethyltransferase gene, glyA) in a single reaction, directly from clinical isolates and human feces. The analytical specificity of the assay was demonstrated with a diverse range of Campylobacter species, related organisms, and other diarrhea-inducing bacterial pathogens. The analytical sensitivity of the multiplexed, PCR assay was 10 genome copies for both C. jejuni and C. coli. Following a rapid DNA extraction method (QIAGEN QIAamp DNA stool Mini Kit), the multiplexed PCR identified C. jejuni or C. coli in 100% of fecal samples containing 10(3) colony-forming units (CFU) per gram of feces. This assay represents the first real-time PCR method capable of detecting and differentiating C. jejuni and C. coli in a single reaction.

Base Sequence↗
PubMed2004

A multiplexed PCR-coupled liquid bead array for the simultaneous detection of four biothreat agents.

We have developed a 10-plexed PCR assay coupled to a 12-plexed liquid bead array to rapidly screen environmental samples for B. anthracis, Y. pestis, F. tularensis, and B. melitensis. Highly validated species-specific primer sets were used to simultaneously amplify multiple diagnostic regions unique to each individual pathogen. Resolution of the mix of amplified products was achieved by PCR product hybridization to corresponding probe sequences, attached to unique sets of fluorescent beads. The hybridized beads were processed through a flow cytometer, which detected presence and quantity of each PCR product. The assay was optimized to allow for maximum sensitivity in a multiplexed format. A high-throughput demonstration was performed where 384 simulated environmental samples were spiked with different amounts of B. thuringensis spores and pathogen DNA. The samples were robotically processed to extract DNA and arrayed for multiplexed PCR-liquid bead detection. The assay correctly identified the presence or absence of each pathogen and collected over 3000 individual data points within a single 8-h shift for approximately $4.00 material costs per environmental sample in a 10-plexed assay.

Bacillus anthracis↗