High-throughput Multiplex Assay Research Articles

P143 High throughput multiplex assay identifies clinically meaningful clusters in systemic sclerosis patients with low cardiovascular risk and no history of heart disease

Abstract Background/Aims People with systemic sclerosis (SSc) often show subclinical evidence of primary heart involvement (pHI), which when clinically evident, is a prognostic factor of poor outcome. We aimed to profile serum proteins in patients with SSc and no history of pHI and identify clinically meaningful clusters to aid early identification and stratification for pHI. Methods Serum from 78 patients from CONVAS (‘CONnective Tissue Disease and VASculitis Cohort’) and ELCASA (‘ELectrophysiology and CArdiac imaging in SclerodermA’) cohorts [no history of pHI, pulmonary hypertension, diabetes or > 2 traditional cardiovascular (CV) risk factors] was used to measure 355 proteins across inflammation, cardiometabolic and cardiovascular II/III Olink panels. Unsupervised hierarchical clustering using scaled normalised protein expression (NPX) was used to define clusters. Demographic and clinical characteristics were compared between clusters and significant differences reported using Kruskal-Wallis rank sum test for continuous or Pearson’s chi-squared test for categorical variables, at the 5% significance threshold. Results Clinical characteristics of the patient cohorts have been previously published (also summarised in table 1,column 2). Unsupervised hierarchical clustering revealed three patient clusters - C1 [27/78 (35%)], C2 [33/78 (42%)] and C3 [18/78 (23%)] with intermediate, low and high protein expression respectively. C2 patients were younger (median age 51 years) compared to C1 (61 years) and C3 (58 years) (p < 0.001). High expression cluster C3 had a significantly higher proportion of males [7/18 (39%)](p = 0.009), shorter disease duration [median 1.15 years, IQR (0.66 to 7.23)], higher modified Rodnan skin scores (MRSS) [median 5.50, IQR (3.25 to 11)] (p = 0.002) and current disease modifying anti-rheumatic drugs (DMARD) use [16/18 (89%)](p = 0.005). NTproBNP and CRP were elevated in C1 and C3, and high-sensitivity troponin I (hsTnI) was highest in C3[median 6.15 IQR (5.08 to 89.58)](p = 0.002); n = 3 within C1 showed especially high NTproBNP (median 227.50pg/ml) and CRP (median 13mg/dl). No differences in antibody profiles were noted across the three clusters (table 1). Conclusion Differences in serum protein expression identifies clinically meaningful clusters of SSc patients with elevated acute phase and serum cardiac markers despite low CV risk profile. Future work should assess their utility as prognostic biomarkers of SSc-pHI. Disclosure R. Shukla: None. R. Dumitru: Grants/research support; ACORN charity and Charitable Foundation Fellowship, Leeds Teaching Hospital. B. Erhayiem: None. G. Fent: None. F. Del Galdo: Consultancies; Abbvie, AstraZeneca, Boehringer-Ingelheim, Capella, Chemomab, GSK, Janssen, Mitsubishi-Tanabe. S. Plein: Other; funded by a British Heart Foundation Personal Chair. D. Plant: None. M.H. Buch: Consultancies; AbbVie, Arxx Therapeutics, Boehringer Ingelheim, Galapagos, Gilead, and Pfizer (all paid to host institution).

Impact of Diabetes on Myocardial Fibrosis in Patients With Hypertension: The REMODEL Study.

Compared with patients with hypertension only, those with hypertension and diabetes (HTN/DM) have worse prognosis. We aimed to characterize morphological differences between hypertension and HTN/DM using cardiovascular magnetic resonance; and compare differentially expressed proteins associated with myocardial fibrosis using high throughput multiplex assays. Asymptomatic patients underwent cardiovascular magnetic resonance: 438 patients with hypertension (60±8 years; 59% males) and 167 age- and sex-matched patients with HTN/DM (60±10 years; 64% males). Replacement myocardial fibrosis was defined as nonischemic late gadolinium enhancement on cardiovascular magnetic resonance. Extracellular volume fraction was used as a marker of diffuse myocardial fibrosis. A total of 184 serum proteins (Olink Target Cardiovascular Disease II and III panels) were measured to identify unique signatures associated with myocardial fibrosis in all patients. Despite similar left ventricular mass (P=0.344) and systolic blood pressure (P=0.086), patients with HTN/DM had increased concentricity and worse multidirectional strain (P<0.001 for comparison of all strain measures) compared to hypertension only. Replacement myocardial fibrosis was present in 28% of patients with HTN/DM compared to 16% of those with hypertension (P<0.001). NT-proBNP (N-terminal pro-B-type natriuretic peptide) was the only protein differentially upregulated in hypertension patients with replacement myocardial fibrosis and independently associated with extracellular volume. In patients with HTN/DM, GDF-15 (growth differentiation factor 15) was independently associated with replacement myocardial fibrosis and extracellular volume. Ingenuity Pathway Analysis demonstrated a strong association between increased inflammatory response/immune cell trafficking and myocardial fibrosis in patients with HTN/DM. Adverse cardiac remodeling was observed in patients with HTN/DM. The novel proteomic signatures and associated biological activities of increased immune and inflammatory response may partly explain these observations.

Standardisation and application of a novel multiplex assay for estimating micronutrient status and inflammatory markers in women of Sauria Paharia and Santhal tribes of Jharkhand.

This study aimed to document the method standardisation and assessment of micronutrient and inflammatory markers in women from indigenous tribal communities of Jharkhand using a low-volume, high-throughput assay. This cross-sectional study was done among women of the reproductive age group from Sauria Paharia and Santhal tribal households (HH) in selected villages. Capillary blood samples were collected from the women during a HH survey to estimate ferritin, soluble transferrin receptor, retinol binding protein 4 and inflammatory biomarkers, C-reactive protein (CRP) and α-1-acid glycoprotein (AGP) using a multiplex assay. Vitamin D and Hb were estimated using an LC-MS technique and cyanmethaemoglobin method, respectively. A multiplex Luminex-based method was developed and standardised. The assay was used to estimate biomarkers in samples from 413 women (178 and 235 from Sauria Paharia and Santhal tribes, respectively). Over 51 % of women had raised CRP or AGP levels. Fe status was significantly better in Sauria Paharia compared with the Santhal women. Anaemia prevalence was 72 % among Santhal women. The proportion of women with Fe deficiency increased after adjusting for inflammation. The overall prevalence of vitamin A deficiency and insufficiency was 25 and 34 %, respectively, with similar prevalence in both tribes. All Santhal women had sufficient vitamin D levels, while 25 and 20 % of Sauria Paharia women had insufficient and deficient vitamin D levels, respectively. Our low-volume, high-throughput multiplex assays may provide a feasible approach for assessing nutritional biomarkers in nutritionally vulnerable hard-to-reach communities.

High-throughput multiplex assays with mouse macrophages on pillar plate platforms

It is challenging to rapidly identify immune responses that reflect the state and capability of immune cells due to complex heterogeneity of immune cells and their plasticity to pathogens and modulating molecules. Thus, high-throughput and easy-to-use cell culture and analysis platforms are highly desired for characterizing complex immune responses and elucidating their underlying mechanisms as well. In response to this need, we have developed a micropillar chip and a 384-pillar plate, printed mouse macrophage, RAW 264.7 cell line in alginate on the pillar plate platforms, and established multiplex cell-based assays to rapidly measure cell viability, expression of cell surface markers, and secretion of cytokines upon stimulation with model compound, lipopolysaccharide (LPS), as well as synthetic N-glycan polymers that mimic native glycoconjugates and could bind to lectin receptors on RAW 264.7 cells. Interestingly, changes in RAW 264.7 cell viability, expression levels of cell surface makers, and release of cytokines measured from the pillar plate platforms in the presence and absence of LPS were well correlated with those obtained from their counterpart, the 96-well plate with 2D-cultured macrophages. With this approach, we identified that α2,3-linked N-sialyllactose polymer has significant macrophage modulation activity among the N-glycan polymers tested. Therefore, we successfully demonstrated that our pillar plate platforms with 3D-cultured macrophages can streamline immune cell imaging and analysis in high throughput in response to compound stimulation. We envision that the pillar plate platforms could potentially be used for rapid characterization of immune cell responses and for screening immune cell-modulating molecules.

SARS-CoV-2-Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach Accounting for Accurate Seroprevalence.

BackgroundThe COVID-19 pandemic necessitates better understanding of the kinetics of antibody production induced by infection with SARS-CoV-2. We aimed to develop a high-throughput multiplex assay to detect antibodies to SARS-CoV-2 to assess immunity to the virus in the general population.MethodsSpike protein subunits S1 and receptor binding domain, and nucleoprotein were coupled to microspheres. Sera collected before emergence of SARS-CoV-2 (n = 224) and of non-SARS-CoV-2 influenza-like illness (n = 184), and laboratory-confirmed cases of SARS-CoV-2 infection (n = 115) with various severities of COVID-19 were tested for SARS-CoV-2–specific IgG concentrations.ResultsOur assay discriminated SARS-CoV-2–induced antibodies and those induced by other viruses. The assay specificity was 95.1%–99.0% with sensitivity 83.6%–95.7%. By merging the test results for all 3 antigens a specificity of 100% was achieved with a sensitivity of at least 90%. Hospitalized COVID-19 patients developed higher IgG concentrations and the rate of IgG production increased faster compared to nonhospitalized cases.ConclusionsThe bead-based serological assay for quantitation of SARS-CoV-2–specific antibodies proved to be robust and can be conducted in many laboratories. We demonstrated that testing of antibodies against multiple antigens increases sensitivity and specificity compared to single-antigen–specific IgG determination.

Comparison of the performance of an amplicon sequencing assay based on Oxford Nanopore technology to real-time PCR assays for detecting bacterial biodefense pathogens

BackgroundThe state-of-the-art in nucleic acid based biodetection continues to be polymerase chain reaction (PCR), and many real-time PCR assays targeting biodefense pathogens for biosurveillance are in widespread use. These assays are predominantly singleplex; i.e. one assay tests for the presence of one target, found in a single organism, one sample at a time. Due to the intrinsic limitations of such tests, there exists a critical need for high-throughput multiplex assays to reduce the time and cost incurred when screening multiple targets, in multiple pathogens, and in multiple samples. Such assays allow users to make an actionable call while maximizing the utility of the small volumes of test samples. Unfortunately, current multiplex real-time PCR assays are limited in the number of targets that can be probed simultaneously due to the availability of fluorescence channels in real-time PCR instruments.ResultsTo address this gap, we developed a pipeline in which the amplicons produced by a 14-plex end-point PCR assay using spiked samples were subsequently sequenced using Nanopore technology. We used bar codes to sequence multiple samples simultaneously, leading to the generation and subsequent analysis of sequence data resulting from a short sequencing run time (< 10 min). We compared the limits of detection (LoD) of real-time PCR assays to Oxford Nanopore Technologies (ONT)-based amplicon sequencing and estimated the sample-to-answer time needed for this approach. Overall, LoDs determined from the first 10 min of sequencing data were at least one to two orders of magnitude lower than real-time PCR. Given enough time, the amplicon sequencing approach is approximately 100 times more sensitive than real-time PCR, with detection of amplicon specific reads even at the lowest tested spiking concentration (around 2.5–50 Colony Forming Units (CFU)/ml).ConclusionsBased on these results, we propose amplicon sequencing assay as a viable alternative to replace the current real-time PCR based singleplex assays for higher throughput biodefense applications. We note, however, that targeted amplicon specific reads were not detectable even at the highest tested spike concentrations (2.5 X 104–5.0 X105 CFU/ml) without an initial amplification step, indicating that PCR is still necessary when utilizing this protocol.

Development and Validation of a Multiplex Non-HLA Antibody Assay for the Screening of Kidney Transplant Recipients.

The best treatment for patients with end-stage renal disease is kidney transplantation. Although graft survival rates have improved in the last decades, patients still may lose their grafts partly due to the detrimental effects of donor-specific antibodies (DSA) against human leukocyte antigens (HLA) and to a lesser extent also by antibodies directed against non-HLA antigens expressed on the donor endothelium. Assays to detect anti-HLA antibodies are already in use for many years and have been proven useful for transplant risk stratification. Currently, there is a need for assays to additionally detect multiple non-HLA antibodies simultaneously in order to study their clinical relevance in solid organ transplantation. This study describes the development, technical details and validation of a high-throughput multiplex assay for the detection of antibodies against 14 non-HLA antigens coupled directly to MagPlex microspheres or indirectly via a HaloTag. The non-HLA antigens have been selected based on a literature search in patients with kidney disease or following transplantation. Due to the flexibility of the assay, this approach can be used to include alternative antigens and can also be used for screening of other organ transplant recipients, such as heart and lung.

Molecular Subtyping of Salmonella Typhimurium with Multiplex Oligonucleotide Ligation-PCR (MOL-PCR).

A multiplex oligonucleotide ligation-PCR (MOL-PCR) assay is a valuable high-throughput technique for the detection of bacteria and viruses, for characterization of pathogens and for diagnosis of genetic diseases, as it allows one to combine different types of molecular markers in a high-throughput multiplex assay. A MOL-PCR assay starts with a multiplex oligonucleotide ligation reaction for detection of the molecular marker, followed by a singleplex PCR for signal amplification and analysis of the MOL-PCR products on a Luminex platform. This last step occurs through a liquid bead suspension array in which the MOL-PCR products are hybridized to MagPlex-TAG beads.In this chapter, we describe the complete procedure for a MOL-PCR assay for subtyping of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant S. 1,4[5],12:i:- from DNA isolation through heat lysis up to data interpretation through a Gödel Prime Product. The subtyping assay consists of 50 discriminative molecular markers and two internal positive control markers divided over three MOL-PCR assays.

Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates.

Increases in expression of ADAM10 and ADAM17 genes and proteins have been evaluated, but not validated as cancer biomarkers. Specific enzyme activities better reflect enzyme cellular functions, and might be better biomarkers than enzyme genes or proteins. However, no high throughput assay is available to test this possibility. Recent studies have developed the high throughput real-time proteolytic activity matrix analysis (PrAMA) that integrates the enzymatic processing of multiple enzyme substrates with mathematical-modeling computation. The original PrAMA measures with significant accuracy the activities of individual metalloproteinases expressed on live cells. To make the biomarker assay usable in clinical practice, we modified PrAMA by testing enzymatic activities in cell and tissue lysates supplemented with broad-spectrum non-MP enzyme inhibitors, and by maximizing the assay specificity using systematic mathematical-modeling analyses. The modified PrAMA accurately measured the absence and decreases of ADAM10 sheddase activity (ADAM10sa) and ADAM17sa in ADAM10-/- and ADAM17-/- mouse embryonic fibroblasts (MEFs), and ADAM10- and ADAM17-siRNA transfected human cancer cells, respectively. It also measured the restoration and inhibition of ADAM10sa in ADAM10-cDNA-transfected ADAM10-/- MEFs and GI254023X-treated human cancer cell and tissue lysates, respectively. Additionally, the modified PrAMA simultaneously quantified with significant accuracy ADAM10sa and ADAM17sa in multiple human tumor specimens, and showed the essential characteristics of a robust high throughput multiplex assay that could be broadly used in biomarker studies. Selectively measuring specific enzyme activities, this new clinically applicable assay is potentially superior to the standard protein- and gene-expression assays that do not distinguish active and inactive enzyme forms.

Abstract 4713: High-throughput multiplex Sequenom MassARRAY clinical diagnostic assay for the identification of actionable genetic variants in hematologic malignancies

Abstract Recent cancer genome sequencing efforts have led to an enhanced understanding of the somatic mutation profile of hematologic malignancies in the context of known cytogenetic abnormalities. For cytogenetically normal acute myeloid leukemia (AML), mutations in NPM1, DNMT3A, CEBPA, TET2 and IDH1/2 are thought to have potential predictive utility and may impact patient management with regard to initial and consolidation therapy, including stem cell transplantation. As most potentially clinically relevant variants currently are not tested for within the diagnostic setting, we established a high-throughput multiplex assay capable of simultaneous detection of a range of somatic mutations in hematologic malignancies. Using iPlex chemistry and the Sequenom MassARRAY platform, we established the Princess Margaret Cancer Centre (PM) Hematologic Malignancies Panel v1.0, comprised of 110 individual assays profiling 186 mutations in 22 relevant genes, in a 16-well assay format. We conducted an in silico analysis to identify genes with hotspot mutations capable of being analyzed by Sequenom; genes with mutations distributed throughout the coding sequence (e.g., CEBPA, TET2) were excluded from the panel. To best integrate this technology in the workflow of our routine clinical molecular diagnostic testing we developed the PM Panel as an RNA-based test, requiring 1 μg of patient RNA for the initial reverse transcription reaction. To validate the assay we tested 222 patient-derived samples, including 5 normal samples, 82 AML (57 with normal cytogenetics, 24 with cytogenetic changes and 1 therapy-related), 44 myelodysplastic syndromes, 32 myeloproliferative neoplasms, 21 additional cases tested for KIT mutation, and 38 B-lymphoid malignancies (including 3 multiple myeloma cases),. Concordance with established lab assays for NPM1, FLT3-TKD and JAK2 mutations was 100%; other identified mutations were verified by Sanger sequencing at 98% concordance (2% of cases were below the limit of sensitivity of Sanger technology). Approximately 50% of samples were positive for at least one mutation with 174 mutations detected overall. Cytogenetically normal AMLs and myelodysplastic syndromes were most informative, with 2.3 and 1.8 mutations per positive case respectively. Samples already known to carry a leukemogenic driver fusion protein (e.g., PML-RARα, AML1-ETO, or BCR-ABL) had a significantly decreased identified somatic mutation load than their cytogenetically normal counterparts (average of 1.4 mutations per case, compared to 0.3, p &amp;lt; 10^-6). The PM Hematologic Malignancies Panel v1.0 is now being integrated into the current clinical diagnostic workflow. We therefore report the development of a versatile, RNA-based high-throughput multiplex platform for the identification of somatic mutations present in hematologic malignancies for use in a clinical diagnostic setting. Citation Format: Mahadeo A. Sukhai, Mariam Thomas, Tong Zhang, Cuihong Wei, Suzanne Trudel, Karen Yee, Mark D. Minden, Andre Schuh, Tracy Stockley, Suzanne Kamel-Reid. High-throughput multiplex Sequenom MassARRAY clinical diagnostic assay for the identification of actionable genetic variants in hematologic malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4713. doi:10.1158/1538-7445.AM2014-4713

Profiles of acute cytokine and antibody responses in patients infected with avian influenza A H7N9.

The influenza A H7N9 virus outbreak in Eastern China in the spring of 2013 represented a novel, emerging avian influenza transmission to humans. While clinical and microbiological features of H7N9 infection have been reported in the literature, the current study investigated acute cytokine and antibody responses in acute H7N9 infection. Between March 27, 2013 and April 23, 2013, six patients with confirmed H7N9 influenza infection were admitted to Drum Tower Hospital, Nanjing, China. Acute phase serum cytokine profiles were determined using a high-throughput multiplex assay. Daily H7 hemagglutinin (HA)-specific IgG, IgM, and IgA responses were monitored by ELISA. Neutralizing antibodies specific for H7N9 viruses were determined against a pseudotyped virus expressing the novel H7 subtype HA antigen. Five cytokines (IL-6, IP-10, IL-10, IFNγ, and TNFα) were significantly elevated in H7N9-infected patients when compared to healthy volunteers. Serum H7 HA-specific IgG, as well as IgM and IgA responses, were detected within 8 days of disease onset and increased in a similar pattern during acute infection. Neutralizing antibodies developed shortly after the appearance of binding antibody responses and showed similar kinetics as a fraction of the total H7 HA-specific IgG responses. H7N9 infection resulted in hallmark serum cytokine increases, which correlated with fever and disease persistence. The novel finding of simultaneous development of IgG, IgM, and IgA responses in acute H7N9 infection points to the potential for live influenza viruses to elicit fast and potent protective antibodies to limit the infection.

A bead-based multiplex assay for the detection of DNA viruses infecting laboratory rodents.

The Federation of European Laboratory Animal Science Association (FELASA) recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals’ physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF) for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.

Chronic unpredictable stress regulates visceral adipocyte-mediated glucose metabolism and inflammatory circuits in male rats

Chronic psychological stress is a prominent risk factor involved in the pathogenesis of many complex diseases, including major depression, obesity, and type II diabetes. Visceral adipose tissue is a key endocrine organ involved in the regulation of insulin action and an important component in the development of insulin resistance. Here, we examined for the first time the changes on visceral adipose tissue physiology and on adipocyte‐associated insulin sensitivity and function after chronic unpredictable stress in rats. Male rats were subjected to chronic unpredictable stress for 35 days. Total body and visceral fat was measured. Cytokines and activated intracellular kinase levels were determined using high‐throughput multiplex assays. Adipocyte function was assessed via tritiated glucose uptake assay. Stressed rats showed no weight gain, and their fat/lean mass ratio increased dramatically compared to control animals. Stressed rats had significantly higher mesenteric fat content and epididymal fat pad weight and demonstrated reduced serum glucose clearing capacity following glucose challenge. Alterations in fat depot size were mainly due to changes in adipocyte numbers and not size. High‐throughput molecular screening in adipocytes isolated from stressed rats revealed activation of intracellular inflammatory, glucose metabolism, and MAPK networks compared to controls, as well as significantly reduced glucose uptake capacity in response to insulin stimulation. Our study identifies the adipocyte as a key regulator of the effects of chronic stress on insulin resistance, and glucose metabolism, with important ramifications in the pathophysiology of several stress‐related disease states.

Multi-analyte suspension arrays for the detection of common viruses: how viable are these assays in clinical laboratories?

Evaluation of: Yu D, Wu S, Wang B, Chen Y, Li L. Rapid detection of common viruses using multi-analyte suspension arrays. J. Virol. Methods 177(1), 64–70 (2011).This article assesses the viability of a recently described multi-analyte suspension array for the detection of herpes simplex viruses-1 and -2, cytomegalovirus, Epstein–Barr virus, human papillomavirus and hepatitis B virus. This methodology was identified by the authors as a means of providing rapid, high-throughput multiplex assays that were easy to use. When paired with PCR assays, multi-analyte suspension arrays have the ability to overcome drawbacks associated with conventional detection methods such as long turnaround time, detection sensitivity and the ability to detect only one pathogen in each round of testing. However, the assays described in this article are still hampered by some key issues including limit of detection, the fact that median fluorescence intensity is not truly a quantitative diagnostic method, and that open molecular diagnostic systems can lead to contamination and/or increased operator-based errors. Although modern pressures on clinical virology laboratories have increased the need to develop a system that can detect pathogens in multiplexed assays, in the future these assays will only become more clinically relevant if they are designed with greater stakeholder input.

A High-Throughput Multiplex Method Adapted for GMO Detection

A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

Interactive Fluorophore and Quencher Pairs for Labeling Fluorescent Nucleic Acid Hybridization Probes

The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.