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PubMed2006

Structural mapping of immunoglobulin subclasses using multiplexed bead flow cytometry.

Monoclonal hybridomas secrete immunoglobulin molecules with a single specificity and distinct class/subclass structure. The determination of immunoglobulin structure can be used to facilitate hybridoma colony management and predict monoclonality. In this report, we used multiplexed bead flow cytometry to define hybridoma class/subclass. The assay was sufficiently sensitive to detect 50 ng/mL of antibody. The multiplexed bead assay efficiently defined traditional class/subclass determinants as well as more subtle patterns of crossreactivity. Further, the assay was combined with Poisson statistics to provide a numerical estimate of hybridoma monoclonality. The sensitivity and flexibility of this approach suggests the utility of multiplexed bead flow cytometry in the early management of immunoglobulin-secreting hybridomas.

Animals↗
PubMed1993

Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs.

Traditionally, DNA used for PCR-based diagnostic analysis has originated from white cells fractionated from whole blood. Although this method yields substantial quantities of DNA, there are some drawbacks to the procedure, including the inconvenience of drawing blood, risk of exposure to blood-borne pathogens, liquid sample handling, and the somewhat involved extraction procedure. Alternatively, DNA for genetic diagnosis has been derived from finger stick blood samples, hair roots, cheek scrapings, and urine samples. Oral saline rinses have also been used extensively as a means of collecting buccal epithelial cells as a DNA source. However, this method still requires liquid sample handling. Herein, we present our results involving the rapid extraction of DNA from buccal cells collected on cytology brushes and swabs for use in PCR reactions, specifically the multiplex amplification of 5 exons within the CFTR gene. The quality of DNA isolated from buccal cells, collected in this manner, has been sufficient to reproducibly support multiplex amplification. Cheek cell samples and the DNA prepared from them as described here are highly stable. The success rate of PCR amplification on DNA prepared from buccal cells is 99%. In a blind study comparing the analysis of 12 mutations responsible for cystic fibrosis in multiplex products amplified with DNA from both blood and buccal cell samples from 464 individuals, there was 100% correlation of results for blood and cheek cell DNA, validating the use of DNA extracted from cheek cells collected on cytology brushes for use in genetic testing.

Base Sequence↗
PubMed2000

Multiplex PCR assays for the detection of clinically relevant antibiotic resistance genes in staphylococci isolated from patients infected after cardiac surgery. The ESPRIT Trial.

Multiresistant staphylococci (82 Staphylococcus aureus and 114 coagulase-negative staphylococci) were characterized by testing with rapid multiplex polymerase chain reaction (PCR) assays for species identification and detection of associated antibiotic resistance genes. These 196 staphylococci were isolated from 149 adult patients who developed wound infection after elective coronary artery bypass grafts and/or valve surgery. The multiplex PCR assays allowed identification of the most common staphylococcal species with S. aureus- and Staphylococcus epidermidis-specific primers as well as the detection of the erythromycin resistance genes ermA, ermB, ermC and msrA, the aminoglycoside resistance gene aac(6')-aph(2"), the oxacillin resistance gene mecA and the penicillin resistance gene blaZ. There was a very good correlation between the genotypic analysis by PCR and the phenotype determined by standard methods of susceptibility testing and identification of staphylococcal species: 100% for erythromycin resistance, 98.0% for gentamicin resistance, 99.0% for oxacillin resistance, 100% for penicillin resistance and 100% for S. aureus and S. epidermidis species identification. This study suggests that the incidence and distribution of the tested clinically relevant antibiotic resistance genes in staphylococci associated with infections after cardiac surgery do not differ from those in strains from other infections. These multiplex PCR assays may be used as diagnostic tools to replace or complement standard methods of susceptibility testing and identification of staphylococci.

Adult↗
PubMedDate not supplied

Multiplex assay of amphetamine, methamphetamine, and ecstasy drug using CEDIA technology.

Using Microgenics unique technology, cloned enzyme donor immunoassay (CEDIA), the multiplex assay (CEDIA Amphetamines/Ecstasy) has been developed for the detection of amphetamine, methamphetamine, and ecstasy drugs at cutoff level either of 500 ng/mL or 1000 ng/mL applicable for either qualitative screening or semiquantitative measurement. The multiplex assay detects the total concentration of amphetamine, methamphetamine, and ecstasy drugs in urine samples. In addition, the assay detects metabolites of parent drugs and structurally related drugs including d,l-amphetamine (67.2%), d,l-methamphetamine (58.4%), MDA (113%), MDMA (199%), MDEA (207%), MBDB (123%), BDB (72%), PMA (24%), and PMMA (100%). The assay is highly specific and exhibits minimal cross-reactivity with the undesirable, structurally related over-the-counter amphetaminelike drugs. Imprecision results (n = 120) demonstrate an intra-assay < 6.3% CV and an interassay < 9.2% CV as performed by a modified NCCLS protocol. Using the SAMHSA mandatory guidelines of a 500-ng/mL cutoff for the immunoassay and a 250-ng/mL cutoff for GC-MS, the assay detects 164 out of 165 GC-MS-positive samples. For drug abuse testing, the multiplex assay presents an excellent method for the detection of multiple drugs with greater sensitivity and broader detection.

Amphetamine↗
PubMed2000

Multiplex PCR of polymorphic markers flanking the CFTR gene; a general approach for preimplantation genetic diagnosis of cystic fibrosis.

Cystic fibrosis (CF) is the first monogenic disorder for which single cell preimplantation genetic diagnosis (PGD) has been successfully applied. The spectrum of mutations in CF is extremely heterogeneous, and hence, the development of mutation-specific PGD protocols is impracticable. The current study reports the development and evaluation of a general multiplex marker polymerase chain reaction (PCR) protocol for PGD of CF. Four closely linked highly polymorphic (CA)(n) repeat markers D7S523, D7S486, D7S480 and D7S490, flanking the cystic fibrosis transmembrane regulator (CFTR) gene, were used. In 99% of the single cells tested (100 leukocytes and 50 blastomeres), multiplex PCR results were obtained and the overall allelic drop out (ADO) rate varied from 2 to 5%. After validation for the presence of ADO and additional alleles, 95% of the multiplex PCR results were accepted to construct the marker genotypes. Depending on the genotype of the couple, and taking into account the embryos lost for transfer due to validation criteria (5%), ADO (0-2%) and single recombination (1.1-3%), in general >90% of the embryos could be reliably genotyped by PGD using a single blastomere. The risk of misdiagnosis equals the chance of a double recombination between informative flanking markers and is <0.05%. Therefore, this polymorphic and multi-allelic marker system is a reliable and generally applicable alternative for mutation-directed PGD protocols. Furthermore, it provides a test for the origin of the detected genotype and also gives an indication of the chromosomal ploidy status of the blastomere tested.

Blastomeres↗
PubMed2002

Genetic follow-up of male offspring born by ICSI, using a multiplex fluorescent PCR-based test for Yq deletions.

De-novo deletions involving AZFa, b, c and d are one of the most common chromosomal aberrations in man resulting in defective spermatogenesis and male infertility. Currently, Yq deletion screening involves either single or multiplex PCR using Y-specific sequence tagged site markers and the subsequent analysis of the amplification products on ethidium bromide-stained agarose gels. To improve the practicality of routine and high throughput Yq testing, we have developed a more sensitive multiplex fluorescent (FL)-PCR screening system using genomic DNA extracted from cheek buccal cells as a readily available PCR template. For genetic follow-up studies of ICSI-conceived children, we also developed a DNA fingerprinting system based on the co-amplification of four highly polymorphic markers to validate family samples and detect any potential extraneous DNA contamination that could cause a misdiagnosis. Multiplex FL-PCR analysis of buccal cell DNA from two infertile men who conceived three sons by ICSI demonstrated that their Yq deletions were vertically transmitted. Fine mapping with additional Yq markers revealed identical deletion endpoints involving the loss of AZFdc sequences. This firstly indicates that the extent of the Yq deletion was unchanged on ICSI transmission and secondly supports the view that AZFdc deletions may arise by a common de-novo event. Analysis of paternal, maternal and sibling DNA fingerprints showed the co-inheritance of parental alleles by each male child and confirmed the expected relationship between each family member. The application of these new FL-PCR based screening tests in genetic follow-up studies will assist in confirming transmission of specific genetic defects to male offspring conceived by ICSI and provide a basis for genetic counselling and potential treatment options as these boys approach sexual maturity.

Adult↗
PubMed1991

Enzymatic multiplex DNA sequencing.

The problem of reading DNA sequence films has been reformulated using an easily implemented, multiplex version of enzymatic DNA sequencing. By utilizing a uniquely tagged primer for each base-specific sequencing reaction, the four reactions can be pooled and electrophoresed in a single lane. This approach has been previously proposed for use with fluorescently labelled probes (1), and is analogous to the principle used in four-dye fluorescence sequencing except that the signals are resolved following electrophoresis (2). After transfer to a nylon membrane, images are obtained separately for each of the four reactions by hybridization using oligonucleotide probes. The images can then be superimposed to reconstitute a complete sequence pattern. In this way the correction of gel distortion effects and accurate band registration are considerably simplified, as each of the four base-specific ladders require very similar corrections. The methods therefore provide the basis for a second generation of more accurate and reliable film reading programs, as well as being useful for conventional multiplex sequencing. Unlike the original multiplex protocol (3), the approach described is suitable for small projects, as multiple cloning vectors are not used. Although more than one vector can be utilized, only a library of fragments cloned into any single phage, phagemid or plasmid vector is actually required, together with a set of tagged oligonucleotide primers.

Base Sequence↗
PubMed1994

Multiplex strand displacement amplification (SDA) and detection of DNA sequences from Mycobacterium tuberculosis and other mycobacteria.

Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying a DNA target sequence prior to detection [Walker et al (1992) Nucleic Acids Res., 20, 1691-1693]. Here we describe a multiplex form of SDA that allows two target sequences and an internal amplification control to be co-amplified by a single pair of primers after common priming sequences are spontaneously appended to the ends of target fragments. Multiplex SDA operates at a single temperature, under the same simple protocol previously developed for single-target SDA. We applied multiplex SDA to co-amplification of a target sequence (IS6110) that is specific to members of the Mycobacterium tuberculosis-complex and a target (16S ribosomal gene) that is common to most clinically relevant species of mycobacteria. Both targets are amplified 10(8)-fold during a 2 hour, single temperature incubation. The relative sensitivity of the system was evaluated across a number of clinically relevant mycobacteria and checked for crossreactivity against organisms that are closely related to mycobacteria.

Base Sequence↗
PubMed2006

Multiplex sequencing of paired-end ditags (MS-PET): a strategy for the ultra-high-throughput analysis of transcriptomes and genomes.

The paired-end ditagging (PET) technique has been shown to be efficient and accurate for large-scale transcriptome and genome analysis. However, as with other DNA tag-based sequencing strategies, it is constrained by the current efficiency of Sanger technology. A recently developed multiplex sequencing method (454-sequencing) using picolitre-scale reactions has achieved a remarkable advance in efficiency, but suffers from short-read lengths, and a lack of paired-end information. To further enhance the efficiency of PET analysis and at the same time overcome the drawbacks of the new sequencing method, we coupled multiplex sequencing with paired-end ditagging (MS-PET) using modified PET procedures to simultaneously sequence 200,000 to 300,000 dimerized PET (diPET) templates, with an output of nearly half-a-million PET sequences in a single 4 h machine run. We demonstrate the utility and robustness of MS-PET by analyzing the transcriptome of human breast carcinoma cells, and by mapping p53 binding sites in the genome of human colorectal carcinoma cells. This combined sequencing strategy achieved an approximate 100-fold efficiency increase over the current standard for PET analysis, and furthermore enables the short-read-length multiplex sequencing procedure to acquire paired-end information from large DNA fragments.

Binding Sites↗
PubMed2003

Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR.

SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye and is included in many commercially available kits at undisclosed concentrations. Binding of SG to double-stranded DNA is non-specific and additional testing, such as DNA melting curve analysis, is required to confirm the generation of a specific amplicon. The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis because the melting temperature (T(m)) of the specific amplicon is analogous to the detection of an electrophoretic band. When using SG for real-time PCR multiplex reactions, discrimination of amplicons should be possible, provided the T(m) values are sufficiently different. Real-time multiplex assays for Vibrio cholerae and Legionella pneumophila using commercially available kits and in-house SG mastermixes have highlighted variability in performance characteristics, in particular the detection of only a single product as assessed by T(m) analysis but multiple products as assessed by agarose gel electrophoresis. The detected T(m) corresponds to the amplicon with the higher G+C% and larger size, suggesting preferential binding of SG during PCR and resulting in the failure to detect multiple amplicons in multiplex reactions when the amount of SG present is limiting. This has implications for the design and routine application of diagnostic real-time PCR assays employing SG.

Bacterial Proteins↗
PubMed2004

SNPWave: a flexible multiplexed SNP genotyping technology.

Scalable multiplexed amplification technologies are needed for cost-effective large-scale genotyping of genetic markers such as single nucleotide polymorphisms (SNPs). We present SNPWave, a novel SNP genotyping technology to detect various subsets of sequences in a flexible fashion in a fixed detection format. SNPWave is based on highly multiplexed ligation, followed by amplification of up to 20 ligated probes in a single PCR. Depending on the multiplexing level of the ligation reaction, the latter employs selective amplification using the amplified fragment length polymorphism (AFLP) technology. Detection of SNPWave reaction products is based on size separation on a sequencing instrument with multiple fluorescence labels and short run times. The SNPWave technique is illustrated by a 100-plex genotyping assay for Arabidopsis, a 40-plex assay for tomato and a 10-plex assay for Caenorhabditis elegans, detected on the MegaBACE 1000 capillary sequencer.

Alleles↗
PubMed1997

Expression of keratins (K10 and K17) in steatocystoma multiplex, eruptive vellus hair cysts, and epidermoid and trichilemmal cysts.

We compared the patterns of keratin 10 (K10) and keratin 17 (K17) expression in epidermoid cysts, trichilemmal cysts, eruptive vellus hair cysts, and steatocystoma multiplex. Epidermoid cysts expressed K10 and eruptive vellus hair cysts expressed K17, whereas trichilemmal cysts and steatocystoma multiplex showed expression of both K10 and K17. Our findings support the opinion that eruptive vellus hair cysts, which stained negative for K10, and steatocystoma multiplex are distinct entities and not variants of one disorder.

Cysts↗
PubMed1988

Steatocystoma multiplex with bilateral preauricular sinuses in four generations.

A rare case of steatocystoma multiplex and bilateral preauricular sinuses in four generations is presented along with a review of the literature. The cause, pathogenesis, and treatment of steatocystoma multiplex is discussed. Steatocystoma multiplex (SCM) is a rare clinical disorder characterized by numerous recurrent cutaneous cysts. Since the first case described by Bosellini in 1898, both nonfamilial and autosomal dominant inheritance patterns have been described. There have been at least 13 well-documented nonfamilial cases. However, since Noonjin and Reynolds in 1948 first described the autosomal dominant inheritance pattern, only 4 well-documented familial cases have been reported. To date, there has been one case report of SCM in four generations, and that case was not associated with any other anomaly. The present case describes the occurrence of SCM in association with bilateral preauricular sinuses in multiple generations.

Adult↗
PubMed2000

The ascertainment of multiplex schizophrenia pedigrees from Daghestan genetic isolates (Northern Caucasus, Russia).

This article describes the preliminary ascertainment of multiplex schizophrenia pedigrees from the isolated mountain region of Daghestan (Northern Caucasus, Russia). Daghestan has a population of two million people and contains 26 aboriginal ethnic groups. Many of the ethnic groups reside in remote mountain villages that can be classified as 'primary isolates'. Prolonged reproductive isolation and severe environmental conditions in the highlands have created diverse, genetically isolated ethnic populations in Daghestan. A number of the isolates in this region contain large extended multiplex schizophrenia pedigrees that are ideal for genetic analyses. During summer expeditions of 1996 and 1997, 14 separate large multiplex schizophrenia pedigrees were ascertained from 14 different mountain villages. Of the 14 kindreds, one had 50 schizophrenic cases available for ascertainment, one had 32, and another had 24. Seven of the remaining pedigrees had between 11 and 23 living cases. Within the kindreds, the number of males with chronic schizophrenia was at least twice that of females. The average age of onset of schizophrenia is 21.2 years for offspring of consanguineous marriages and 17.4 years for offspring of nonconsanguineous marriages (P = 0.033). Although the pedigrees ascertained from the remote mountain villages may not be representative of the general population, they are unique kindreds for mapping schizophrenia susceptibility genes.

Ethnicity↗
PubMed2005

Multiplexed analysis of serum cytokines as biomarkers in squamous cell carcinoma of the head and neck patients.

OBJECTIVE: Our objective was to characterize cytokine profiles in the systemic circulation of patients with active squamous cell carcinoma of the head and neck (SCCHN) compared with long-term tobacco smokers as controls. Furthermore, we hypothesized that persistent immune dysregulation in patients cured of their disease may be reflected in altered cytokine profiles in the systemic circulation. STUDY DESIGN: Retrospective, case-control study. METHODS: Using three well-defined clinical cohorts of SCCHN patients and smoker controls, we analyzed 100 microL of serum for a panel of 10 cytokines to determine whether serum cytokine profiles could distinguish clinically defined groups of SCCHN patients. Statistical analysis of multiplexed cytokine profiles was applied to classify three clinically defined groups: active SCCHN patients, treated SCCHN patients with no evidence of disease for over 3 years, and matched disease-free controls. Discrimination of outcome status was accomplished using classification trees, and 10-fold cross-validation was implemented to assess classification accuracy using independent data. RESULTS: We show that multiplexed cytokine and chemokine profiling may be performed to reflect the immune status of SCCHN patients. Selected cytokine profiles indicate that immunologic responses to carcinogenesis may not normalize even in the absence of tumor for over 3 years. CONCLUSION: Multiplexed serum cytokine profiles may be applicable to early detection, for screening those at high risk for SCCHN, and as clinically predictive biomarkers of disease status in successfully treated patients.

Biomarkers↗
PubMed1998

Application of multiplex PCR using species-specific primers within the 16S rRNA gene for rapid identification of Nocardioides strains.

For the rapid identification of Nocardioides strains, multiplex PCR, using 16S rDNA as target gene, was used and its value was evaluated. Forward primers specific for Nocardioides albus, Nocardioides jensenii, Nocardioides plantarum and Nocardiodes simplex, among the five validly described Nocardioides species, were designed from the alignment of 165S rDNA sequences. Nocardioides luteus has been shown to be a member of the same species as N. albus by recent molecular systematic studies and preliminary DNA-DNA relatedness tests. Therefore, N. albus and N. luteus were considered as members of the same species in this study. Each primer was found to be species-specific by specificity testing. N. albus NSP01T, N. jensenii NSP19T, N. plantarum NSP21T and N. simplex NSP22T could be clearly differentiated by PCR products characteristics for each species in the multiplex PCR assays. N. luteus gave an identical results to N. albus NSP01. The additional 17 strains of N. albus and the additional four stains of N. simplex gave PCR products identical to those of N. albus NSP01T and N. simplex NSP22T, respectively. Multiplex PCR was found to be rapid, species-specific and reproducible. The technique evaluated in this study proved to be effective for rapidly identifying Nocardioides strains to species level.

Actinomycetales↗
PubMed2005

Rapid and simple detection of blaCTX-M genes by multiplex PCR assay.

A novel multiplex PCR assay is described (CTX-Mplex PCR) that allows rapid detection of bla(CTX-M) genes and discrimination between groups 1, 2, 9 and 25/26. The specificity and sensitivity of the assay were evaluated with 10 control strains and then applied to 62 clinical isolates. The multiplex PCR detected and classified bla(CTX-M) genes with 100 % accuracy. The utilization of a denaturing HPLC WAVE system to size the PCR products automatically from the multiplex PCR enhances the assay by saving time and costs.

Bacterial Proteins↗
PubMed1992

Detection of the most common mutations causing beta-thalassemia in Mediterraneans using a multiplex amplification refractory mutation system (MARMS).

A rapid, simple, cost-effective, non-radioactive method for detection of the most common mutations causing beta-thalassemia in Mediterranean people has been developed by combining multiplexing with the amplification refractory system. This approach, the multiplex amplification refractory mutation system (MARMS), provides an easy assay for direct detection of normal and mutant beta-globin genes in homozygotes and heterozygotes. The strategy involves multiplex PCR of four of the five regions of interest within the beta-globin gene in a single reaction containing a common oligoprimer and either the normal or mutant oligonucleotides corresponding to IVS-1 nucleotide 1 or IVS-1 nucleotide 6, IVS-1 nucleotide 110, codon 39, and IVS-2 nucleotide 1 regions. Primers are chosen so that the sizes of the four PCR products differ, thereby facilitating detection on agarose gels following amplification. Patient samples are primed with either four normal or four mutant oligonucleotide mixtures and the common oligoprimer, and PCR products run in parallel on gels to detect band presence or absence. This approach simplifies mutation detection and shows promise for automation employing fluorescent-tagged primers.

Alleles↗