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PubMed2002

Fluorescent multicolor multiplex homogeneous assay for the simultaneous analysis of the two most common hemochromatosis mutations.

We report the development of a qualitative fluorescent multiplex homogeneous assay designed for the detection of the two most common hemochromatosis mutations using dual-labeled fluorescent probes. The assay is able to detect four allelic variants in a single closed tube using a single thermocycling protocol. The procedure combines the great sensitivity of the polymerase chain reaction, the specificity provided by allele-specific oligonucleotide hybridization using the 5(') nuclease assay format, and the higher throughput of a multicolor fluorescence detection procedure. Genomic DNA was prepared from whole blood specimens using standard procedures. Following DNA sample preparation, two regions of the hemochromatosis gene (HFE) including the H63D and C282Y mutations were coamplified and detected in real-time by four different fluorescently labeled allele-specific oligonucleotide probes. Assay specificity was demonstrated by a blind methods comparison study that included 37 DNA samples from individuals with a known HFE genotype. Results from the study showed that the multicolor multiplex HFE assay unambiguously classified all possible genotypes for the HFE gene C282Y and H63D mutations(1). This technique will be useful for research and molecular diagnostic laboratories and can be easily adapted for the detection of other single nucleotide polymorphisms.

Alleles↗
PubMed2005

Comparison of three in-house multiplex PCR assays for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis using real-time and conventional detection methodologies.

AIMS: To develop and evaluate multiplex PCR assays for Chlamydia trachomatis and Neisseria gonorrhoeae, using real-time and conventional PCR detection methodologies. METHODS: Two real-time multiplex PCR assays, using nuclease (TaqMan-ABI7500) and hybridisation (LightCycler) probe formats, and a third assay using conventional PCR with solid-phase hybridisation and colour detection, were developed. The porA pseudogene was targeted for N. gonorrhoeae, and the major outer membrane protein gene for C. trachomatis. A total of 145 urogenital specimens were tested in all assays, and the results were compared with the Roche Cobas Amplicor assay. RESULTS: There was little difference in clinical sensitivity and specificity, result discrimination and test cost for the three in-house assays. Our results showed that competitive inhibition of the PCR occurred in some samples that were positive for both organisms. CONCLUSIONS: These results highlight the suitability and versatility of three multiplex PCR methods for the detection of C. trachomatis and N. gonorrhoeae.

Adolescent↗
PubMed2006

Development of a sensitive and specific assay combining multiplex PCR and DNA microarray primer extension to detect high-risk mucosal human papillomavirus types.

The importance of assays for the detection and typing of human papillomaviruses (HPVs) in clinical and epidemiological studies has been well demonstrated. Several accurate methods for HPV detection and typing have been developed. However, comparative studies showed that several assays have different sensitivities for the detection of specific HPV types, particularly in the case of multiple infections. Here, we describe a novel one-shot method for the detection and typing of 19 mucosal high-risk (HR) HPV types (types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82). This assay combines two different techniques: multiplex PCR with HPV type-specific primers for amplification of viral DNA and array primer extension (APEX) for typing. This novel method has been validated with artificial mixtures of HPV DNAs and clinical samples that were already analyzed for the presence of mucosal HPV types by a different consensus PCR method, i.e., GP5+/GP6+. Our data showed a very good agreement between the results from the multiplex PCR/APEX assay and those from the GP5+/GP6+ PCR (overall rates of HPV positivity, 63.0 and 60.9%, respectively). Whereas the GP5+/GP6+ PCR was slightly more sensitive for the detection of HPV type 16 (HPV-16), multiplex PCR-APEX found a higher number of infections with HPV-33, HPV-53, and multiple HPV types. These favorable features and the high-throughput potential make our present novel assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of mucosal HR HPV types in cervical specimens.

DNA Primers↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed2003

Performance of the APTIMA Combo 2 assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in female urine and endocervical swab specimens.

The greater sensitivity of nucleic acid amplification tests (NAATs) for Chlamydia trachomatis and Neisseria gonorrhoeae permits the use of urine and other noninvasive specimens, which can increase the reach and decrease the costs of public health screening programs aimed at controlling these infections. This study evaluated the performance of the APTIMA Combo 2 assay, a multiplex assay based on the transcription-mediated amplification reaction, for the simultaneous detection of both pathogens in endocervical swab and urine specimens from females. Combo 2 assay results were compared with patient infected status, which were available by using other commercial NAATs. Sensitivity and specificity for C. trachomatis were 94.2 and 97.6%, respectively, in swabs and 94.7 and 98.9%, respectively, in first-catch urine (FCU). Sensitivity and specificity for N. gonorrhoeae were 99.2 and 98.7%, respectively, in swabs and 91.3 and 99.3%, respectively, in FCU. The assay reliably detected both infections in coinfected patients. The Combo 2 assay can be recommended for use with endocervical swab and urine specimens from females, especially for screening tests for asymptomatic women in sexually transmitted disease surveillance programs. This Food and Drug Administration-cleared assay can be a useful tool in efforts to reduce the prevalence and incidence of C. trachomatis and N. gonorrhoeae infections in sexually active women and to prevent their costly and serious sequelae.

Cervix Uteri↗
PubMed2026

'Truthsets' for clinical validation of large-scale functional assays: Practice recommendations from Cancer Variant Interpretation Group UK (CanVIG-UK).

BACKGROUND: Large-scale functional assays, including multiplex assays of variant effect, have substantial potential to resolve variants of uncertain significance (VUS), particularly for rare missense variants where clinical and population evidence are limited. The ClinGen assay-level clinical validation framework described by Brnich et al provided baseline guidance for the use of functional data for variant classification. However, clear consensus regarding construction of variant 'truthsets' by which to clinically validate functional data remains lacking. METHODS: CanVIG-UK developed consensus recommendations for truthset construction through an iterative national consultation process involving the CanVIG Steering Advisory Group (CStAG), wider CanVIG-UK membership, and engagement with international functional genomics experts. Consultation was based on previous analyses of 2,120 truthset constructions examining the impact of truthset composition on evidence point allocation within the ClinGen assay-level clinical validation framework. RESULTS: Across several consultations, CanVIG-UK established nine guiding principles and seven best-practice recommendations for assay-level clinical validation, using the assumed context of an assay for a cancer susceptibility gene where loss-of-function is the mechanism of pathogenicity. The principal recommendation stipulates, where assays are intended for use in interpretation of largely missense variants, the truthset used to validate should comprise only missense variants. Rather than mixtures of different variant types which may serve to over-estimate assay performance. Additional recommendations support option for relaxation of truthset stringency to improve power, augmentation of benign missense truthsets with systematically derived 'proxy-clinical' benign variants, independent clinical validation separate from assayist-defined validation, and careful evaluation of missense score distributions against that of protein-truncating and synonymous variants. Guidance is also provided for scenarios with limited pathogenic truthset availability and for assays reporting multiple deleterious zones or readouts. CONCLUSIONS: The CanVIG-UK principles and recommendations for truthset construction upon the ClinGen assay-level clinical validation framework, while aiming to form a baseline for future discussion regarding other functional and disease contexts and helping to address the gap between publication of new data and routine clinical implementation.

Journal Article↗
PubMed2001

Multiplexed enzyme assays in capillary electrophoretic single-use microfluidic devices.

We describe a method of performing multiple enzyme assays in a single reaction vessel. The resolving power of capillary electrophoresis enables several enzyme assays to be analyzed at high speed in microfluidic arrays. Multiplexed measurement can increase throughput significantly without requiring highly dense microfluidic arrays. Enzyme assays in a multiplexed format for selected kinases in this work show essentially identical performance to assays performed individually. This establishes an approach for screening one compound against multiple enzyme targets simultaneously. Another potential application for performing multiplexed enzyme assay is to study protein-protein (especially enzyme-enzyme) interaction by monitoring the enzymatic activity changes.

Amino Acid Sequence↗
PubMed2003

A multiplex PCR assay for the simultaneous detection and discrimination of the seven Eimeria species that infect domestic fowl.

This study reports the development of a novel multiplex PCR assay based on SCAR (Sequence-Characterised Amplified Region) markers for the simultaneous diagnosis of the 7 Eimeria species that infect domestic fowl. Primer pairs specific for each species were designed in order to generate a ladder of amplification products ranging from 200 to 811 bp. Sensitivity tests for each species were carried out, showing a detection threshold of 1-5 pg, which corresponds approximately to 2-8 sporulated oocysts. Distinct isolates of the 7 Eimeria species from different geographical sources were tested and successfully detected by the assay. All the species were amplified homogeneously, whether or not one of them was present in a high quantity, indicating that there was no cross-interference. The assay was also tested with different sources of Taq DNA polymerase and thermocycler models, confirming the high reproducibility of the reaction. The economy of consumables and labour represented by a single-tube reaction greatly facilitates the molecular diagnosis of a large number of samples, making it appropriate for field epizootiological surveys. We propose the use of this multiplex PCR assay as a rapid and cost-effective diagnostic method for the detection and discrimination of the 7 Eimeria species that infect domestic fowl.

Animals↗
PubMed2005

A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus.

A multiplex PCR assay using three collagenase-targeted primer pairs for the species-specific detection of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus was developed. The results highlight the species specificity of the three primer sets designed. Because of the increasing importance of Vibrio spp. in human foodborne diseases, molecular approaches for routine microbial screening and monitoring of clinical, environmental, and food samples also have become more important. The results of this study indicate that the gene coding for collagenase should be used as an alternative molecular target to discriminate among the three Vibrio species.

DNA Primers↗
PubMed1996

Multiplex PCR assay for the routine detection of Listeria in food.

The development and validation of a multiplex PCR assay for the detection of Listeria that can be employed in routine investigation of food samples are described. The assay, which employs a short culture enrichment step followed by isolation of bacterial cells and detection by multiplex PCR reaction, is highly sensitive and specific for the detection of Listeria monocytogenes and all other Listeria species. Over 350 food samples were tested in parallel by standard cultural procedures and the PCR assay, with no false-positive or false-negative results obtained with the PCR assay. Compared to the standard cultural methods the PCR assay is highly sensitive, cost effective and extremely rapid with results obtained within 48 h from sample receipt.

Food Microbiology↗
PubMed2001

Flow cytometric microsphere-based immunoassay: analysis of secreted cytokines in whole-blood samples from asthmatics.

The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microsphere-based assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-gamma), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data (r > 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 (P = 0.003) and less IFN-gamma (P = 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-gamma than that of atopic nonasthmatic subjects (P = 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases.

Adult↗
PubMed2001

[Rapid detection of enterohemorrhagic Escherichia coli in food with multiplex PCR assay].

Rapid detection of enterohemorrhagic Escherichia coli (EHEC) in food was achieved by using multiplex PCR assay. The assay was designed to amplify the 1109, 302 and 228 bp regions of corresponding virulence genes eaeA, hlyAB, slt1 and/or slt2, by using specific primers for each one reaction. In 60 EHEC and other strains, the above three virulence gene sequences were co-amplified from DNA isolated from 12 EHEC O157:H7 strains, 1 O26:H11 strain and 1 O111:H8 strain; eaeA sequence was amplified from DNA isolated from 2 EAEC strains; slt1/2 sequence was amplified from DNA isolated from 1 VTEC strain. No specific amplification product was produced from other 43 non-EHEC strains. For the detection of EHEC in food, results could be obtained within 8 hrs, and the sensitivity of assay was less than or equal to 1.6 cfu/g(ml) of food. The method was applied to 126 food samples, and EHEC was detected in 3 samples.

Animals↗
PubMed1997

Multiplex PCR assay for immediate identification of the infecting species in patients with mycobacterial disease.

Rapid identification of infecting mycobacterial species enables appropriate medical care decisions to be made. Our aim was to demonstrate the clinical usefulness of the multiplex PCR assay, a test based on PCR, which permits direct identification of 12 mycobacterial species in clinical specimens. A total of 259 specimens from 177 patients who had clinical symptoms of mycobacterial disease but for whom there were difficulties in diagnosis were tested. Specimens were analyzed within 48 h of receipt of the sample. Mycobacteria were identified in 102 specimens; 66 specimens contained nontuberculous mycobacteria, and 36 specimens contained Mycobacterium tuberculosis complex mycobacteria. The PCR assay identified the mycobacterial species in 43 (97.7%) of 44 microscopy- and culture-positive specimens and in 15 (93.8%) of 16 culture-positive, microscopy-negative specimens. It also permitted species identification in infections caused by more than one mycobacterial species. For 56 (96.5%) of the 58 specimens from patients with infections caused by opportunistic mycobacteria, the organisms were identified with the PCR assay. The test was useful also for the identification of fastidious mycobacteria, e.g., M. genavense, and those that cannot be cultured, e.g., M. leprae. After resolution of discrepant results, the sensitivity of the PCR assay was 97.9%, the specificity was 96.9%, the positive predictive value was 95.0%, and the negative predictive value was 98.7%. For culture these values were 60.8, 100, 100, and 81.0%, respectively. Thus, the multiplex PCR assay enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.

AIDS-Related Opportunistic Infections↗
PubMed2000

Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA.

Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.

Bacterial Typing Techniques↗
PubMed2004

Multiplex PCR assay for detection of bacterial pathogens associated with warm-water Streptococcosis in fish.

A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens involved in warm-water streptococcosis in fish (Streptococcus iniae, Streptococcus difficilis, Streptococcus parauberis, and Lactococcus garvieae). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The sensitivity of the multiplex PCR using purified DNA was 25 pg for S. iniae, 12.5 pg for S. difficilis, 50 pg for S. parauberis, and 30 pg for L. garvieae. The multiplex PCR assay was useful for the specific detection of the four species of bacteria not only in pure culture but also in inoculated fish tissue homogenates and naturally infected fish. Therefore, this method could be a useful alternative to the culture-based method for the routine diagnosis of warm-water streptococcal infections in fish.

Animals↗
PubMed2004

Evaluation of multiplexed fluorescent microsphere immunoassay for detection of autoantibodies to nuclear antigens.

Antibodies to extractable nuclear antigens (ENA) are found in a variety of collagen vascular diseases. Determining the individual specificities of these antibodies is extremely useful in establishing the disease diagnosis and in some cases the prognosis. With a multiplexed fluorescent microsphere immunoassay, reactivity to five of the most diagnostically useful ENA was measured in 249 serum samples, including samples from 56 patients previously documented to have systemic lupus erythematosus (SLE). Results of the multiplexed assay were compared to results from established ENA enzyme-linked immunosorbent assays (ELISAs), and the agreement, sensitivity, and specificity, respectively, for the five ENA evaluated were as follows: SSA, 99.1, 100.0, and 98.8%; SSB, 98.6, 88.9, and 99.5%; Sm, 97.6, 95.8, and 97.9%; RNP, 97.2, 92.7, and 98.8%; Scl-70, 93.6, 50.0, and 99.0%. In the 56 confirmed SLE patients, the frequency of significant concentrations of autoantibodies with the multiplexed assay was 21.4% for SSA, 7.1% for SSB, 10.7% for Sm, 32.1% for RNP, and 0% for Scl-70. The new flow cytometric bead-based multiplexed assay showed excellent correlation with the well-established single-analyte ELISA methods for four of five the ENA markers investigated in this study. The most notable discrepancies between the two assays were for the Scl-70 antigen, which was most often resolved in favor of the multiplexed assay. Our studies show that the multiplexed microsphere-based immunoassay is a sensitive and specific method for the detection and semiquantitation of ENA antibodies in human sera.

Antibodies, Antinuclear↗
PubMed2004

High-throughput Y-STR typing of U.S. populations with 27 regions of the Y chromosome using two multiplex PCR assays.

Two Y-chromosome short tandem repeat (STR) multiplex polymerase chain reaction (PCR) assays were used to generate haplotypes for 19 single copy and 3 multi-copy Y-STRs. A total of 27 PCR products were examined in each sample using the following loci: DYS19, DYS385 a/b, DYS388, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS426, DYS437, DYS438, DYS439, DYS447, DYS448, DYS450, DYS456, DYS458, DYS460, DYS464 a/b/c/d, H4, and YCAII a/b. The first multiplex is the Y-STR 20plex previously described by Butler et al. [Forensic Sci. Int. 129 (2002) 10]. The second multiplex is a novel Y-STR 11plex and includes DYS385 a/b, DYS447, DYS448 and the new markers DYS450, DYS456, DYS458, and DYS464 a/b/c/d. These two multiplexes were tested on 647 males from three United States population sample sets: 260 African Americans, 244 Caucasians, and 143 Hispanics. Haplotype comparisons between common loci included in the 20plex and 11plex assays as well as commercially available kits found excellent agreement across a sampling of the population samples. The multi-copy loci DYS464, DYS385, and YCAII were the most polymorphic followed by the following single copy Y-STRs: DYS458, DYS390, DYS447, DYS389II, DYS448, and DYS456. Samples containing the most common type in the European database could be well resolved with additional markers beyond the minimal haplotype loci.

Alleles↗