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PubMed2002

Multiplex detection of herpesviruses in tear fluid using the "stair primers" PCR method: prospective study of 93 patients.

Human herpesviruses can infect the eye and be excreted subsequently in tears. The aim of the present study was to use a multiplex PCR to detect herpesviruses (HSV-1, -2, VZV, CMV, EBV, HHV-6) in tears from normal subjects and from patients with pathological conditions (acute herpes, zoster, papillary conjunctivitis, and dry eye). Schirmer test strips were used to collect tear fluid from 93 patients, sampling both eyes. DNA was then extracted from the 186 samples by chromatography, and viral DNA amplified using a commercialised multiplex "stair primer" method. Thirty-four samples (18.3%) contained Taq inhibitors. The multiplex test gave positive results for HSV and VZV in tear fluid from patients with acute dendritic keratitis (3 patients) and acute ocular zoster (4 patients) and was, therefore, considered effective in testing samples from patients with acute lesions. HSV-1 and HSV-2 were found in two samples from patients with metaherpetic corneal scarring. Among 28 cases of dry eye, two were positive for HHV-6, the latter being associated with EBV in one patient. HHV-6 was also found in 4 out of 54 cases of papillary conjunctivitis. This raised occurrence of HHV-6 in dry eye or papillary conjunctivitis, suggests new clinical patterns for HHV-6 latency or reactivation. Detection of EBV in 1 out of 80 healthy eyes confirms previous evidence that lacrimal glands constitute potentially a site for latent-phase EBV.

Conjunctivitis, Viral↗
PubMed2002

Detection and typing of respiratory adenoviruses in a single-tube multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) assay that is capable of detecting and typing six serotypes of respiratory adenovirus (Ad) was developed, using multiple sets of type-specific primers. The detection of each different serotype depended on distinguishing different numbers and sizes of amplification products on agarose gels following PCR. The multiplex PCR was tested with 26 clinical Ad isolates and other respiratory viruses including influenza viruses, parainfluenza viruses, and respiratory syncytial viruses as well as respiratory bacterial pathogens such as Chlamydia pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae. The multiplex PCR for the detection and typing of Ads gave an excellent correlation with the results by conventional typing with type-specific antisera. This assay may serve as a rapid means of confirming Ad with simultaneous serotype identification of the isolates. It will also have relevance as an adjunctive tool to conventional serotyping for diagnostic and epidemiological purposes.

Adenovirus Infections, Human↗
PubMed2003

Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results.

Multiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three "y" or "b" fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform-ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.

Algorithms↗
PubMed2006

Multiplex detection of surface molecules on colorectal cancers.

A technique of fluorescence multiplexing is described for analysis of the plasma membrane proteome of colorectal cancer cells from surgically resected specimens, enabling detection and immunophenotyping when the cancer cells are in the minority. A single-cell suspension was prepared from a colorectal tumour, and the mixed population of cells was captured on a CD antibody microarray. The cancer cells were detected using a fluorescently tagged antibody for carcinoembryonic antigen (CEA-Alexa647) or epithelial cell adhesion marker (EpCAM-Alexa488). Using this multiplexing procedure, dot patterns from colorectal cancers were distinct from those of adjacent normal tissue. Subtraction of the expression levels for each antigen from normal tissue from those for the cancer shows differential expression in the cancer of CD66c, CD15s, CD55, CD45, CD71, CD45RO, CD11b and CEA, in descending order. Cells captured on the same microarray were also labelled with fluorescent CD3-phycoerythrin antibody revealing the presence of tumour-infiltrating lymphocytes. The immunophenotypes of T lymphocytes from the tumour samples showed differential expression of HLA-DR, TCR alpha/beta, CD49d, CD52, CD49e, CD5, CD95, CD28, CD38 and CD71, in descending order. Fluorescence multiplexing of mixed cell populations captured on a single antibody microarray enables expression profiling of multiple sub-populations of cells within a tumour sample.

CD4 Antigens↗
PubMed2004

Multiplexed hybridizations of positively charge-tagged peptide nucleic acids detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Peptide nucleic acid (PNA) is a novel class of DNA analogues in which the entire sugar-phosphate backbone is replaced by a pseudopeptide counterpart. Owing to its neutral character and the consequent lack of electrostatic repulsion, PNA exhibits very stable heteroduplex formation with complementary nucleic acid that is essentially ionic strength independent and enables hybridization under minimum salt conditions. This feature as well as its superior ion stability and easy ionization compared to DNA renders PNA very attractive for hybridization-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) applications. We have developed an approach to DNA characterization that takes advantage of multiplexed PNA hybridizations analyzed by MALDI-TOFMS. Our motivation was the further development of oligonucleotide fingerprinting, an efficient technique for cDNA and genomic DNA library characterization. Through positive 'charge-tagging' of PNA the efficiency of detection in MALDI-TOFMS was considerably enhanced permitting an unparalleled degree of multiplexing. Results from the simultaneous hybridization of 21 charge-tagged PNA hexamer oligonucleotides showed that genomic DNA and cDNA clones are successfully characterized on the basis of their hybridization profiles. The degree of multiplexing achieved may render a significant increase in throughput and hence efficiency of oligonucleotide fingerprinting possible.

DNA, Complementary↗
PubMed2006

Multiplex PCR-based detection of potential fumonisin-producing Fusarium in traditional African vegetables.

Culture-independent methods employed in fungal genetic studies using in vitro amplification (PCR) and analysis of specific genes or gene fragments have proved to be useful for detection, identification, and molecular taxonomy of various plant pathogens including Fusarium spp. This approach may be faster than culture-dependent methods, and could especially be of value for the rapid detection of slow-growing toxin-producing species in food samples. The present study was aimed at the development and evaluation of multiplex PCR-based methods for the detection and identification of potential fumonisin-producing Fusarium spp. in traditional morogo-leafy vegetables supplementing the maize-based staple diet of rural communities in South Africa. In these rural subsistence settings, some morogo plants grow as weeds in maize fields where they might become contaminated with potential fumonisin-producing Fusarium strains before being collected for consumption. Substances released by senescent vegetables could induce toxin production during storage. Using fumonisin-positive MRC Fusarium verticillioides strains as reference cultures, the following primer sets for the detection of specific gene fragments in fumonisin-positive Fusarium spp. were evaluated: (i) the conserved transcription elongation factor gene (EF-1), (ii) the FUM1 gene encoding polyketide synthase for fumonisin B1 production, and (iii) 18S rRNA gene. Preliminary results indicated that, these DNA fragments were amplified from MRC reference strains as well as Fusarium spp. isolated from morogo. The annealing temperature for the multiplex PCR was 55 degrees C and each reaction contained 25 pmol of each of the primer sets EF and FUM1 and 12.5 pmol of the 18S primer set. The detection limit of the individual primers was up to 1 ng and for the multiplex up to 10 ng. This demonstrates the potential of this method for the detection of potential fumonisin-positive strains.

Fumonisins↗
PubMed1999

A generic particle-based nonradioactive homogeneous multiplex method for high-throughput screening using microvolume fluorimetry.

We have developed a novel fluorescence-based homogeneous binding assay for high-throughput screening of chemical compounds. In this assay, a Cy5- or Cy5.5-labeled ligand binds to receptor immobilized on a particle, either a bead or a cell. The resulting localized signal can be detected by a modified microvolume fluorimeter (MVF). When a molecule which competes with the labeled ligand is present, the localized fluorescence on cells or beads is reduced. Image processing software enumerates events and analyzes fluorescence intensity. We describe MVF assays for the IL-1 and IL-5 receptors. Using synthetic peptides with a range of affinities for the IL-1 receptor, we obtained IC(50) data consistent with those determined by radioligand binding assays. Because the image processing software can discriminate among events with different diameters, we were able to develop a multiplex assay, in which the IL-1R and IL-5R assays were carried out in the same well with each receptor immobilized on a different size of bead. IC(50) values generated in the multiplex assay for ligands specific to each receptor were comparable to those determined independently. Finally, similar IC(50) values were obtained in a 16-microl volume in an 864-well plate. This homogeneous, nonradioactive, miniaturizable, and multiplex-capable assay holds much promise for screening of combinatorial libraries and compound collections.

Animals↗
PubMed1999

Color multiplexing hybridization probes using the apolipoprotein E locus as a model system for genotyping.

Fluorescent hybridization probes were multiplexed for color genotyping of the apolipoprotein E locus using model oligonucleotide targets. Fluorescence resonance energy transfer was observed during adjacent hybridization of 3'-fluorescein-labeled "donor" probes paired with 5'-labeled "acceptor" probes with different emission spectra reporting at codons 112 and 158. The acceptor dyes emitted at either 640 nm (LightCycler Red 640) or 705 nm (LightCycler Red 705) and were monitored with a LightCycler, a thermal cycler with an integrated fluorimeter. The color of the acceptor dye identified each site and the characteristic melting temperatures of the fluorescein-labeled probes identified single base changes within each codon. Color compensation of temperature-dependent spectral overlap was applied to completely separate each channel. Competition between the probes and the complementary strand for the target sequence decreased resonance energy transfer, indicating an advantage of single-stranded target. Hybridization probes of the same length, but different GC content are T(m) shifted by the same amount during A:C mismatch duplex melting. Genotyping was optimal at both sites if melting curve analysis was preceded by a slow (1 degrees C/s) annealing phase. Although each site preferred different concentrations of Mg(2+) and target strand for optimal genotyping, conditions for multiplexing were found. This method, along with an appropriate amplification technique, should allow real-time multiplex genotyping from genomic DNA.

Apolipoproteins E↗
PubMed1999

Contig assembly of bacterial artificial chromosome clones through multiplexed fluorescence-labeled fingerprinting.

A rapid multiplexed fingerprinting method has been developed for bacterial artificial chromosome (BAC) contig assembly. Defined subsets of BAC DNA fragments that result from digestion by three paired restriction endonucleases are labeled with unique fluorescent F-ddATP for each subset. Lists of the labeled fragment size are generated by an ABI 377 DNA sequencer and the GeneScan analysis software and then processed by an assembly program, FPC (Fingerprinted Contigs), to produce contig maps. Data obtained from the multiplexed labeling permit detection of smaller overlaps than is observed when data from a single double-digest are analyzed. The method has been tested on 98 BACs from chromosome 22 regions where large-scale sequencing is under way and also through simulation, using randomly generated BAC clones derived from existing DNA sequence data. In each case, contig assembly results demonstrated the advantages of multiplexed fingerprinting.

Chromosomes, Bacterial↗
PubMed1998

Controlled multiplex PCR of enterotoxigenic Clostridium perfringens strains in food samples.

A controlled multiplex polymerase chain reaction (PCR) for the detection of Clostridium(C.) perfringens enterotoxin gene (cpe) was established and compared with an in vitro antigenic detection method. Thecpe PCR and the classical method of electric immunodiffusion gave identical results. The predicted specific amplicon of the cpe gene was generated from all of the tested enterotoxigenic C. perfringens strains. In contrast, cultures of any other Clostridium species tested by PCR were negative (100% sensitivity, 100% specificity). Addition of an alphatoxin (plc) gene specific PCR as an in process control reaction was performed in order to prevent false negative PCR results. The PCR detection limit was 0.5 ng of genomic C. perfringens DNA per ml of bouillon culture. By contaminating minced meat with C. perfringens reference strains, the multiplex PCR was established as a tool for routine diagnostic laboratories. The detection limit was approximately 3.0x10(5)C. perfringens cells per gram meat. The results demonstrate the multiplex PCR as an easy, specific, sensitive and time saving diagnostic procedure. Application of this improved method should enhance the knowledge concerning epidemiological aspects of food borne diseases caused by enterotoxigenic C. perfringens.

Clostridium perfringens↗
PubMed1999

A multiplex PCR for Massachusetts and Arkansas serotypes of infectious bronchitis virus.

Infectious bronchitis virus (IBV), the prototype of the coronavirus family, is an enveloped, single-stranded RNA virus with a genome size of approximately 27.6 kilobase. Infectious bronchitis virus causes an acute, highly contagious respiratory and urogenital disease of chickens which results in significant economic losses in commercial broilers, layers and breeders. A rapid, highly sensitive and specific method is needed in the differential diagnosis of infections of different serotypes. A multiplex polymerase chain reaction (PCR) method was developed and optimized to simultaneously detect Massachusetts (Mass) and Arkansas (Ark) serotypes of IBV. One common primer and two serotype specific primers were chosen from the S1 gene sequences of IBV and used in one PCR reaction. Under optimized PCR conditions, two serotype specific PCR products, 1026 bp for Mass and 896 bp for Ark, respectively, were amplified and detected by agarose gel electrophoreses. The specificity of the technique was verified by using 20 different strains and isolates of IBV, and other avian bacterial and viral pathogens. Using a serial 10-fold dilution of the artificial mixture of both Mass and Ark samples, the detection limit was found to be 5 pg RNA after 35 cycles of PCR. The multiplex PCR was able to detect and differentiate both serotypes in embryonated eggs that were co-infected with different EID50virus titers of Mass 41 and Ark 99. The multiplex PCR developed in this study will be valuable for rapid identification, differential diagnosis, and epidemiological studies of these two serotypes of IBV infections.

Animals↗
PubMed1999

Molecular screening of potential HNPCC patients using a multiplex microsatellite PCR system.

Microsatellite instability (MSI) is a molecular hallmark of the H ereditary N on- P olyposis C olorectal C ancer (HNPCC) syndrome occurring in about 80-90% of the tumours and also in sporadic tumours of different organs, albeit at lower frequency. Highly unstable colorectal tumours (MSI-H) have different histopathological features and tend to have a better prognosis compared to neoplasms without (MSS) or with low levels of microsatellite instability (MSI-L). Since MSI classification allows the identification of potential HNPCC patients and might represent a valuable diagnostic parameter an increasing demand for high-throughput microsatellite analysis will arise. Therefore, we have adapted five diagnostic microsatellites, m(odified) ACTC, mBAT26, mD5S107, mD5S406 and mD13S153, to allow coamplification. Using this multiplex polymerase chain reaction (PCR) system 29 colorectal tumour tissues with known MSI status could be unambiguously identified as MSI-H (13 cases) or MSI-L/MSS (16 cases). Highly unstable colorectal tumour detection frequency of individual markers reached 77% (mD5S406), 85% (mACTC), 85% (mD5S107), 92% (D13S153) and 100% (mBAT26) showing similar sensitivity but improved specificity as compared with a microsatellite reference panel. In a prospective analysis of 31 colorectal tumours, the multiplex PCR system identified five MSI-H cases. Multiplex MSI PCR is a time saving and cost-effective method not restricted to specific technical equipment and applicable to a variety of microsatellite-based genotyping approaches.

Colorectal Neoplasms↗
PubMed1999

Simultaneous detection of erythromycin-resistant methylase genes ermA and ermC from Staphylococcus spp. by multiplex-PCR.

A comparative analysis of the two most dominant erythromycin-resistance determinant genes in Staphylococcus sppnamely, the ermA and ermC genes, was carried out. Sixty erythromycin-resistant strains of Staphylococcus spp. were tested, of which 24 were avian and 36 were clinical isolates. Our results indicated the prevalence of ermA over the ermC gene as opposed to the widely held opinion of the ermC gene being the most dominant resistance determinant gene. A multiplex-PCR assay was developed to detect the presence of ermA and ermC genes. Two pairs of primers, specific for the detection of ermA and ermC genes, were used in a multiplex-polymerase chain reaction (PCR) assay to yield amplified DNA products of 610 and 520 bp, respectively. Their digestion with restriction enzyme FokI that yielded a 477 bp and a 132 bp digestion product for ermA and a 333 bp and a 187 bp digestion product for ermC confirmed the authenticity of PCR products. The method could be used to amplify the ermA and ermC genes with as little as 5 pg of template DNA. The use of excess primers or the template DNA resulted in gene-specific amplification and no non-specific amplification was observed by changing the primer to primer or template to primer ratios. Furthermore, no amplification from erythromycin-sensitive S. aureus strain was observed. Using this assay, the poultry strains were found to contain either ermA alone (50%) or a combination of ermA (100%) and ermC (50%) both. The clinical strains contained either ermA (94.5%) or ermC (5.5%) but never both. The gene-specific internal probes were also used to verify the above findings and a high degree of correlation between the multiplex PCR and Southern hybridization data was observed.

Animals↗
PubMed2001

Multiplex PCR for the detection of tetracycline resistant genes.

Specific primer pairs were selected for the PCR amplification of 14 tetracycline resistant genes commonly found in Gram positive and Gram negative organisms. Combinations of primer pairs were used in multiplex PCR reactions to detect specific groups of tet genes as follows; Group I tet (B), tet (C), tet (D); Group II tet (A), tet (E), tet (G); Group III tet (K), tet (L), tet (M), tet (O), tet (S); Group IV tetA (P), tet (Q), tet (X). To test the multiplex PCR, Groups I and II were used on 25 clinical isolates of Salmonella enterica serovar Typhimurium DT104. Group III primers were used to investigate 19 clinical isolates of methicillin-resistant Staphylococcus aureus. Multiplex PCR should result in significant savings in terms of labour and cost in analysis of a large number of strains when compared with using an individual PCR for targeting each gene. It may also be a useful method to differentiate the types of tetracycline resistance when used as an additional marker for the purpose of outbreak investigation and surveillance.

DNA Fingerprinting↗
PubMed1998

Detection of staphylococcal genes directly from cerebrospinal and peritoneal fluid samples using a multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) that can simultaneously detect eubacterial isolates and the methicillin-susceptibility of staphylococcal isolates from cerebrospinal and peritoneal fluid samples was compared to conventional microbiological methods. Using conventional methods, bacteria were isolated from 8% (29/350) of the cerebrospinal fluid samples and from 5% (3/60) of the peritoneal fluid samples. All isolates except two Staphylococcus epidermidis isolates were also detected using the multiplex PCR. Coagulase-negative staphylococci and Staphylococcus aureus were correctly identified using both methods. The multiplex PCR can rapidly and simultaneously detect eubacteria, and the methicillin susceptibility of staphylococci from samples containing > or = 10(2) cfu/ml of bacteria.

Ascitic Fluid↗
PubMed1994

Detection of deletion mutations extending beyond the HPRT gene by multiplex PCR analysis.

A multiplex PCR assay was developed for the rapid analysis of deletion size at the hypoxanthine phosphoribosyltransferase (hprt) locus. The DNA sequence of mapped DNA segments flanking the hprt gene was determined. These cloned DNAs were derived from the ends of a set of overlapping yeast artificial chromosomes (YAC) defining a contig of 8 Mb at Xq26 and including hprt. We used "bubble" PCR to isolate an additional YAC end-clone. Seven primer pairs were derived from DNA sequence analysis of the clones and incorporated into a multiplex PCR assay. These primer pairs define loci located approximately 750 kb and 350 kb upstream of hprt and 300 kb, 540 kb, 900 kb, 1260 kb, and 1400 kb downstream of hprt. A primer pair for an unlinked and unselected gene sequence (K-ras) was also included in the multiplex reaction to serve as an internal positive control. Using this new assay, hprt mutant DNAs can be screened to determine the extent of deletion. Deletions larger than 2 Mb have been identified and show that large deletions can be tolerated at this hemizygous locus.

Adult↗
PubMed2000

SMASH imaging with an eight element multiplexed RF coil array.

SMASH (SiMultaneous Acquisition of Spatial Harmonics) is a technique which can be used to acquire multiple lines of k-space in parallel, by using spatial information from a radiofrequency coil array to perform some of the encoding normally produced by gradients. Using SMASH, imaging speed can be increased up to a maximum acceleration factor equal to the number of coil array elements. This work is a feasibility study which examines the use of SMASH with specialized coil array and data reception hardware to achieve previously unattainable accelerations. An eight element linear SMASH array was designed to operate in conjunction with a time domain multiplexing system to examine the effectiveness of SMASH imaging with as much as eightfold acceleration factors. Time domain multiplexing allowed the multiple independent array elements to be sampled through a standard single-channel receiver. SMASH-reconstructed images using this system were compared with reference images, and signal to noise ratio and reconstruction artifact power were measured as a function of acceleration factor. Results of the imaging experiments showed an almost constant SNR for SMASH acceleration factors of up to eight. Artifact power remained low within this range of acceleration factors. This study demonstrates that efficient SMASH imaging at high acceleration factors is feasible using appropriate hardware, and that time domain multiplexing is a convenient strategy to provide the multiple channels required for rapid imaging with large arrays.

Biophysical Phenomena↗
PubMed2005

A new "miniSTR-multiplex" displaying reduced amplicon lengths for the analysis of degraded DNA.

A multiplex PCR was designed for the loci D2S1338, D16S539, D18S51, TH01 and FGA using redesigned primers in order to reduce the lengths of the amplification products compared to the designs used in commercially available multiplex PCR kits, also including amelogenin. The new PCR primers were used to amplify highly degraded DNA from casework samples, which had shown no or only poor results for these loci in previous analyses with standard primer sets. The application of the new miniSTR-multiplex resulted in an increased overall typing success rate for degraded DNA samples. In a concordance study between the conventional and the newly designed primers, no genotype differences were revealed in 124 randomly selected individuals.

Amelogenin↗