Nucleic acid sample preparation techniques for bead-based suspension arrays.

Multiplexing in biological assays allows the simultaneous detection of multiple analytes in a single reaction, which reduces time, labor, and cost as compared to single reaction-based detection methods. Microsphere- or bead-based suspension array technologies, such as the Luminex® xMAP® system, offer high-throughput detection of nucleic acids through a variety of different assay chemistries. Common with most nucleic acid chemistries, for bead-based or other microarray technologies, is the need for efficient extraction and purification of the nucleic acids from the specimen of interest. Often, the optimal method will be dictated by the requirements of the up-front enzymatic chemistry, such as PCR, primer extension, branched DNA (bDNA), etc. For bead-based microarray platforms, the user must also be cognizant of proteins and other contaminants present in reactions that require heat denaturation, as that can lead to bead aggregation or agglutination, preventing the reading of assay results. This review describes and highlights some of the nucleic acid extraction and purification methods that have been used successfully for bead-based nucleic acid analysis, for both prokaryotic and eucaryotic nucleic acids, from a variety of sample types.

The genesis and evolution of bead-based multiplexing.

Multiplexed analysis has the advantage of allowing for simultaneous detection of multiple analytes in a single reaction vessel which reduces time, labor, and cost as compared to single-reaction-based detection methods. Microsphere-based suspension array technologies, such as the Luminex® xMAP® system, offer high-throughput detection of both protein and nucleic acid targets in multiple assay chemistries. After Luminex's founding in 1995, it quickly became the leader in bead-based multiplexing solutions. Today, xMAP Technology is the most widely adopted bead-based multiplexing platform with over 35,000 peer-reviewed publications, an installed base of approximately 15,500 instruments, and over 70 Luminex Partners offering more than 1300 research use kits as well as custom assay solutions. Because of the open architecture of the xMAP platform it has been implemented in a variety of applications that range from transplant medicine, biomarker discovery and validation, pathogen detection, drug discovery, vaccine development, personalized medicine, neurodegeneration, and cancer research.

Evaluation of the Aries Bordetella Assay for Detection and Identification of Bordetella pertussis in Nasopharyngeal Swab Specimens.

The Aries Bordetella assay (Aries BA) (Luminex Corporation) recently received FDA clearance for the detection and differentiation of Bordetella pertussis and Bordetella parapertussis nucleic acids in nasopharyngeal swab (NPS) samples. The objective of this study was to evaluate the performance of the Aries BA in comparison to that of the BioFire FilmArray respiratory panel (RP). The Aries BA was evaluated using retrospective, remnant nasopharyngeal swabs (NPS), previously tested by FilmArray RP. Performance characteristics evaluated included positive percent agreement (PPA) and negative percent agreement (NPA) with the FilmArray RP. Discordant analysis was performed using bidirectional sequencing. A time and motion study was performed to compare the laboratory workflow of the two tests. Three hundred samples were included in the study. There were no samples positive for B. parapertussis The PPA and NPA of the Aries BA were 61.1% (95% confidence interval [CI], 35.8 to 82.7%) and 100% (95% CI, 98.7 to 100%). Discordant results included five Bordetella bronchiseptica results that were incorrectly identified as B. pertussis by the FilmArray RP and one false-negative result for both the Aries BA and the FilmArray RP. The overall agreement between the Aries BA and FilmArray RP for the detection of B. pertussis was considered good at 97.7% with a kappa value of 0.71 (95% CI, 0.51 to 0.9). The Aries BA offers a new diagnostic option for the rapid and targeted approach to the diagnosis of pertussis. Unlike the FilmArray RP, the Aries BA did not cross-react with B. bronchiseptica in our study, although a larger sample set should be tested to confirm this finding.

Clinical Evaluation of the Luminex NxTAG Respiratory Pathogen Panel.

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.

Use of Luminex MagPlex magnetic microspheres for high-throughput spoligotyping of Mycobacterium tuberculosis isolates in Port-au-Prince, Haiti.

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.

Multiplexed suspension array immunoassays for detection of antibodies to pneumococcal polysaccharide and conjugate vaccines.

Combination and polyvalent vaccines not only provide protection against several different pathogens at the same time but can also increase vaccine protection against pathogens that have closely related pathogenic strains or serotypes. Multiplexed serological testing is a preferred method for determining the efficacy of combination and polyvalent vaccines, as it reduces the need for conducting multiple individual assays to confirm immune responses and cross-reactivity, uses less sample, and can be faster, more reliable, and more cost-effective. Bead-based suspension array technologies, such as the Luminex® xMAP® Technology, are often used for development of multiplexed serological assays for various vaccine trials and for routine testing in clinical laboratories to determine immune status of vaccinated individuals. This article reviews publications describing the development and implementation of bead-based multiplexed serological assays for detection of immune responses to polyvalent polysaccharide and conjugate vaccines against Streptococcus pneumoniae. Many of these serological assays on the bead array platform have been further optimized and expanded over time and are still widely used today.

Diagnosis and Management of Bloodstream Infections With Rapid, Multiplexed Molecular Assays.

Bloodstream infection is a major health concern, responsible for considerable morbidity and mortality across the globe. Prompt identification of the responsible pathogen in the early stages of the disease allows clinicians to implement appropriate antibiotic therapy in a timelier manner. Rapid treatment with the correct antibiotic not only improves the chances of patient survival, but also significantly reduces the length of hospital stay and associated healthcare costs. Although culture has been the gold standard and most common method for diagnosis of bloodstream pathogens, it is being enhanced or supplanted with more advanced methods, including molecular tests that can reduce the turnaround time from several days to a few hours. In this article, we describe two rapid, molecular bloodstream infection panels that identify the most common pathogens and associated genetic determinants of antibiotic resistance - the Luminex® VERIGENE® Gram-Positive Blood Culture Test and the VERIGENE® Gram-Negative Blood Culture Test. We conducted a search on PubMed to retrieve articles describing the performance and impact of these tests in the clinical setting. From a total of 48 articles retrieved, we selected 15 for inclusion in this review based on the type and size of the study and so there would be minimum of three articles describing performance and three articles describing the impact post-implementation for each assay. Here we provide a comprehensive review of these publications illustrating the performance and clinical utility of these assays, demonstrating how genotypic tests can benefit diagnostic and antimicrobial stewardship efforts.

Molecular detection of harmful algal blooms (HABs) using locked nucleic acids and bead array technology.

Harmful algal blooms (HABs) are a serious public health risk in coastal waters. As the intensity and frequency of HABs continue to rise, new methods of detection are needed for reliable identification. Herein, we developed a high-throughput, multiplex, bead array technique for the detection of the dinoflagellates Karenia brevis and Karenia mikimotoi. The method combined the Luminex detection system with two novel technologies: locked nucleic acid-modified oligonucleotides (LNA) and Mirus Label IT(®) nucleic acid technology. To study the feasibility of the method, we evaluated the performance of modified and unmodified LNA probes with amplicon targets that were biotin labeled with two different strategies: direct chemical labeling (Mirus Label IT) versus enzymatic end-labeling (single biotinylated primer). The results illustrated that LNA probes hybridized to complementary single-stranded DNA with better affinity and displayed higher fluorescence intensities than unmodified oligonucleotide DNA probes. The latter effect was more pronounced when the assay was carried out at temperatures above 53°C degree. As opposed to the enzymatic 5' terminal labeling technique, the chemical-labeling method enhanced the level of fluorescence by as much as ~83%. The detection limits of the assay, which were established with LNA probes and Mirus Label IT system, ranged from 0.05 to 46 copies of rRNA. This high-throughput method, which represents the first molecular detection strategy to integrate Luminex technology with LNA probes and Mirus Label IT, can be adapted for the detection of other HABs and is well suited for the monitoring of red tides at pre-blooming and blooming conditions.

Comparison of xMAP Salmonella Serotyping Assay With Traditional Serotyping and Discordance Resolution by Whole Genome Sequencing.

Salmonella spp. are a major cause of foodborne illness throughout the world. Traditional serotyping by antisera agglutination has been used as a standard identification method for many years but newer nucleic acid-based tests have become available that may provide advantages in workflow and test turnaround time. In this study, we evaluated the Luminex® xMAP® Salmonella Serotyping Assay (SSA), a multiplex nucleic acid test capable of identifying 85% of the most common Salmonella serotypes, in comparison to the traditional serum agglutination test (SAT) on 4 standard strains and 255 isolates from human (224), environmental, and food (31) samples. Of the total of 259 isolates, 256 could be typed by the SSA. Of these, 197 (77.0%) were fully typed and 59 (23.0%) were partially typed. By SAT, 246 of the 259 isolates (95%) were successfully typed. Sixty isolates had discrepant results between SAT and SSA and were resolved using whole genome sequencing (WGS). By SAT, 80.0% (48/60) of the isolates were consistent with WGS while by SSA 91.7% (55/60) were partially consistent with WGS. By serovar, all 30 serovars except one tested were fully or partially typable. The workflow comparison showed that SSA provided advantages over SAT with a hands-on time (HOT) of 3.5 min and total turnaround time (TAT) of 6 h, as compared to 1 h HOT and 2-6 days TAT for SAT. Overall, this study showed that molecular serotyping is promising as a rapid method for Salmonella serotyping with good accuracy for typing most common Salmonella serovars circulating in China.

Identification and differentiation of clinically relevant mycobacterium species directly from acid-fast bacillus-positive culture broth.

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.

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.

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

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.

Rapid screening for 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator gene by Lminex 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.

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.

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.

Pharmacogenetics using Luminex® xMAP® technology: a method for developing a custom multiplex single nucleotide polymorphism mutation assay.

Sequence variations in the human genome can affect the development of diseases and provide markers for the identification of genetic diseases and drug susceptibility. Single Nucleotide Polymorphisms (SNPs), the most abundant sequence variations in the genome, are used in pharmacogenetics as indicators of drug therapy efficacy in individuals and are important road maps in the route to personalized medicine. This chapter describes the development of PCR based custom multiplex SNP mutation analysis assays using Luminex(®) Multi-Analyte Profiling (xMAP(®)) Technology. Up to 500 different mutations can be detected in a single well and up to 384 samples can be analyzed per run.

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.

Real-time polymerase chain reaction detection methods.

Real-time Polymerase Chain Reaction (PCR) is a quickly developing technology that has built upon the classic end-point PCR detection methods. In this article, we will review recent patents related to various chemistries used for nucleic acid detection during real-time PCR amplification. Real-time assay chemistries are subdivided into several main categories including DNA-binding agents, molecular beacons, hybridization probes, hydrolysis probes, and dye-primer based systems. Specific advantages and applications of each category are highlighted herein.