Sherry A. Dunbar

Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection.

Abstract Background As we enter the post-genome sequencing era and begin to sift through the enormous amount of genetic information now available, the need for technologies that allow rapid, cost-effective, high-throughput detection of specific nucleic acid sequences becomes apparent. Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can greatly reduce the time, cost and labor associated with single reaction detection technologies. Methods The Luminex® xMAP™ system is a multiplexed microsphere-based suspension array platform capable of analyzing and reporting up to 100 different reactions in a single reaction vessel. This technology provides a new platform for high-throughput nucleic acid detection and is being utilized with increasing frequency. Here we review specific applications of xMAP technology for nucleic acid detection in the areas of single nucleotide polymorphism (SNP) genotyping, genetic disease screening, gene expression profiling, HLA DNA typing and microbial detection. Conclusions These studies demonstrate the speed, efficiency and utility of xMAP technology for simultaneous, rapid, sensitive and specific nucleic acid detection, and its capability to meet the current and future requirements of the molecular laboratory for high-throughput nucleic acid detection.

Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP™ system

Escherichia coli, Salmonella, Listeria monocytogenes and Campylobacter jejuni are bacterial pathogens commonly implicated in foodborne illnesses. Generally used detection methods (i.e., culture, biochemical testing, ELISA and nucleic acid amplification) can be laborious, time-consuming and require multiple tests to detect all of the pathogens. Our objective was to develop rapid assays to simultaneously detect these four organisms through the presence of antigen or DNA using the Luminex LabMAP system. For nucleic acid detection, organism-specific capture probes corresponding to the 23S ribosomal RNA gene (rrl) were coupled covalently to LabMAP microspheres. Target molecules included synthetic complementary oligonucleotides and genomic DNA isolated from ATCC type strains or other well-characterized strains of each organism. Universal PCR primers were designed to amplify variable regions of bacterial 23S ribosomal DNA, yielding biotinylated amplicons of 86 to 109 bp in length. Varying quantities of targets were hybridized to the combined microsphere sets, labeled with streptavidin-R-phycoerythrin and analyzed on the Luminex(100) system. Results of nucleic acid detection assays, obtained in 30 to 40 min following amplification, correctly and specifically identified each bacterial species with a detection sensitivity of 10(3) to 10(5) genome copies. Capture-sandwich immunoassays were developed with organism-specific antibodies coupled to different microsphere sets. Microspheres were incubated with organism-specific standards and reactivity was assessed with biotinylated detection antibodies and streptavidin-R-phycoerythrin. In the immunoassays, microsphere-associated fluorescence was organism concentration dependent with detectable response at < or = 1000 organisms/ml and with no apparent cross-reactivity. We have demonstrated that the Luminex LabMAP system is a rapid, flexible platform capable of simultaneous, sensitive and specific detection of pathogens. The practical significance of this multiplexing approach would be to provide more timely, economical and comprehensive information than is available with conventional isolation and identification methodologies.

Quantitative, multiplexed detection of Salmonella and other pathogens by Luminex xMAP suspension array.

We describe a suspension array hybridization assay for rapid detection and identification of Salmonella and other bacterial pathogens using Luminex xMAP technology. The Luminex xMAP system allows simultaneous detection of up to 100 different targets in a single multiplexed reaction. Included in the method are the procedures for (1) design of species-specific oligonucleotide capture probes and PCR amplification primers, (2) coupling oligonucleotide capture probes to carboxylated microspheres, (3) hybridization of coupled microspheres to oligonucleotide targets, (4) production of targets from DNA samples by PCR amplification, and (5) detection of PCR-amplified targets by direct hybridization to probe-coupled microspheres. The Luminex xMAP suspension array hybridization assay is rapid, requires few sample manipulations, and provides adequate sensitivity and specificity to detect and differentiate Salmonella and nine other test organisms through direct detection of species-specific DNA sequences.

Identification and Differentiation of Clinically Relevant Mycobacterium Species Directly from Acid-Fast Bacillus-Positive Culture Broth

ABSTRACT Mycobacterium species cause a variety of clinical diseases, some of which may be species specific. Therefore, it is clinically desirable to rapidly identify and differentiate mycobacterial isolates to the species level. We developed a rapid and high-throughput system, MycoID, to identify Mycobacterium species directly from acid-fast bacillus (AFB)-positive mycobacterial culture broth. The MycoID system incorporated broad-range PCR followed by suspension array hybridization to identify 17 clinically relevant mycobacterial complexes, groups, and species in one single reaction. We evaluated a total of 271 AFB-positive culture broth specimens, which were identified by reference standard methods in combination with biochemical and molecular tests. The overall identification agreement between the standard and the MycoID system was 89.7% (perfect match) or 97.8% (one match in codetection). In comparison to the standard, the MycoID system possessed an overall sensitivity of 97.1% and specificity of 98.8%. The 159 Mycobacterium avium-M. intracellulare complex isolates were further identified to the species level by MycoID as being M. avium (n = 98; 61.1%), M. intracellulare (n = 57; 35.8%), and mixed M. avium and M. intracellulare (n = 2; 1.3%). M. avium was recovered more frequently from sterile sites than M. intracellulare (odds ratio, 4.6; P = 0.0092). The entire MycoID procedure, including specimen processing, can be completed within 5 h, providing rapid and reliable identification and differentiation of mycobacterium species that is amenable to automation. Additional differentiation of Mycobacterium avium-M. intracellulare complex strains into M. avium and M. intracellulare may provide a tool to better understand the role of Mycobacterium avium-M. intracellulare complex isolates in human disease.

Luminex(®) multiplex bead suspension arrays for the detection and serotyping of Salmonella spp.

In this chapter we describe two commercially available bead-based molecular assays for detection, identification and serotyping of Salmonella. The xTAG(®) Gastrointestinal Pathogen Panel (GPP) is a qualitative multiplex test for the simultaneous detection of nucleic acids from Salmonella plus 14 other gastroenteritis-causing bacteria, viruses, and parasites from stool specimens. xTAG GPP uses the Luminex(®) xTAG universal array technology for the identification of specific target sequences combined with the xMAP(®) bead multiplexing platform for detection of the targets that were present in the starting sample. The xMAP Salmonella Serotyping Assay (SSA) is a multiplex nucleic acid-based direct hybridization assay for molecular identification of the serotype of Salmonella isolates. In xMAP SSA, target sequences amplified from cultured Salmonella isolates are captured by hybridization to sequence-specific capture probes which have been coupled to the multiplexed bead sets. Herein we provide detailed protocols for each of these assays and present data which describe their performance characteristics for detection and serotyping Salmonella.

Rapid Screening for 31 Mutations and Polymorphisms in the Cystic Fibrosis Transmembrane Conductance Regulator Gene by Luminex® xMAP™ Suspension Array

A suspension array hybridization assay is described for the detection of 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using Luminex xMAP technology. The Luminex xMAP system allows simultaneous detection of up to 100 different targets in a single multiplexed reaction. Included in the method are the procedures for design of oligonucleotide capture probes and PCR amplification primers, coupling oligonucleotide capture probes to carboxylated microspheres, hybridization of coupled microspheres to oligonucleotide targets, production of targets from DNA samples by multiplexed PCR amplification, and detection of PCR-amplified targets by direct hybridization to probe-coupled microspheres. Mutation screening with the system is rapid, requires relatively few sample manipulations, and provides adequate resolution to reliably genotype the 25 CFTR mutations and 6 CFTR polymorphisms contained in the ACMG/ACOG/NIH-recommended core mutation panel for general population CF carrier screening.

Use of Luminex MagPlex Magnetic Microspheres for High-Throughput Spoligotyping of Mycobacterium tuberculosis Isolates in Port-au-Prince, Haiti

ABSTRACT Genotyping of Mycobacterium tuberculosis strains became indispensable for understanding tuberculosis transmission dynamics and designing measures to combat the disease. Unfortunately, typing involves sophisticated laboratory analysis, is expensive, and requires a high level of technical expertise, which limited its use in the resource-poor countries where the majority of tuberculosis cases occur. Spoligotyping is a PCR-based M. tuberculosis complex genotyping method with advantages of technical simplicity, numerical output, and high reproducibility. It is based on the presence or absence of 43 distinct “spacers” separating insertion elements in the direct repeat region of the M. tuberculosis genome. The spoligotyping assay involves reverse hybridization of PCR products to the capture spacers attached to nitrocellulose membranes or to microspheres. Here we report modification of the classic 43-spacer method using the new generation of Luminex multiplexing technology with magnetic microspheres. The method was successfully established and validated on strains with known spoligotypes in our laboratory in Haiti. The distribution of spoligotypes determined in a collection of 758 recent M. tuberculosis isolates was in accordance with previous data for Haitian isolates in the SITWITWEB international database, which were obtained with the traditional membrane-based method. In the present form, spoligotyping may be suitable as a high-throughput, first-line tool for genotyping of Mycobacterium tuberculosis in countries with limited resources.

Advanced techniques for detection and identification of microbial agents of gastroenteritis.

Gastroenteritis persists as a worldwide problem, responsible for approximately 2 million deaths annually. Traditional diagnostic methods used in the clinical microbiology laboratory include a myriad of tests, such as culture, microscopy, and immunodiagnostics, which can be labor intensive and suffer from long turnaround times and, in some cases, poor sensitivity. [corrected]. This article reviews recent advances in genomic and proteomic technologies that have been applied to the detection and identification of gastrointestinal pathogens. These methods simplify and speed up the detection of pathogenic microorganisms, and their implementation in the clinical microbiology laboratory has potential to revolutionize the diagnosis of gastroenteritis.

Clinical Evaluation of the Luminex NxTAG Respiratory Pathogen Panel

ABSTRACT An evaluation of the Luminex NxTAG Respiratory Pathogen Panel was performed on 404 clinical respiratory specimens. Clinical sensitivities and specificities of the assay compared to those of the reference methods were 80.0% to 100.0% and 98.9% to 100.0%, respectively. Correct genotyping information was provided for 95.5% of influenza virus A specimens. The closed-tube format of the assay simplified the workflow and minimized carryover contamination.

Microsphere-Based Multiplex Immunoassays: Development and Applications Using Luminex® xMAP® Technology

This chapter describes the Luminex range of bead-based multiplex assays, which are based on flow cytometry. These are widely used by third party manufacturers and individual researchers to develop multiplexed immunoassays for up to 500 different analytes simultaneously. The equipment and reagents available are described and the theory explained. This chapter includes detailed information to enable assays to be developed using the core system, including buffer formulations and their applications. Step-by-step protocols are provided on coupling of proteins to microspheres, confirmation of antibody coupling, and the final immunoassay (immunometric, indirect, competitive and combined immunometric/competitive). Notes are also provided on assay optimization. A list of commercially available products from third-party manufacturers is included.