A simple nucleic acid hybridization/latex agglutination assay for the rapid detection of polymerase chain reaction amplicons

Vollenhofer‐Schrumpf, Sabine; Buresch, Ronald; Schinkinger, Manfred

doi:10.1016/j.mimet.2006.10.017

Cited by 4 publications

(4 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A particle agglutination-based assay is a relatively simple diagnostic method that has been widely applied to detect viruses, blood groups, and DNA hybridization. 35,[37][38][39][40] In this study, streptavidin-coated fluorescent particles (450 nm) were used as probe nanoparticles, while multibiotinylated BSA (bovine serum albumin) was mixed with the particles to induce particle aggregation. Fig.…”

Section: Selective Trapping Of Particles At the 800 Nm Nanoslit Membranementioning

confidence: 99%

Nanoslit membrane-integrated fluidic chip for protein detection based on size-dependent particle trapping

et al. 2014

View full text Add to dashboard Cite

This paper describes the fabrication of a nanoslit membrane-integrated fluidic chip (Nanoslit-Chip) used for trapping and concentrating micro-/nano-particles of desired size and its application in detecting biological molecules based on target-induced particle aggregation. To trap particles of a specific size, a large scale uniform sized nanoslit fluid channel array is fabricated on a silicon dioxide membrane. A small number of fluorescence labeled particles in a large volume of solution are concentrated into a monolayer of particles in a small nanoslit membrane, which enables us to effectively quantify them via fluorescence intensity. In addition, the particles of desired size (1.8 μm) are readily separated from the mixture of particles with a different size (450 nm) in Nanoslit-Chip size, and then quantified via fluorescence measurements. Finally, the Nanoslit-Chip is successfully applied to the sensitive detection of proteins by target-induced particle aggregation, trapping, and quantification. This shows its potential as a biological and clinical device for quantitative and sensitive detection of biological molecules.

show abstract

Section: Selective Trapping Of Particles At the 800 Nm Nanoslit Membranementioning

confidence: 99%

Nanoslit membrane-integrated fluidic chip for protein detection based on size-dependent particle trapping

et al. 2014

View full text Add to dashboard Cite

show abstract

“…There had been some attempts to achieve the multiplexed analysis within the microfluidic devices, for example, chemically functionalized capillaries array assembled into microfluidic chips for chemical-sensing [23], a bead-bed immunoassay system fabricated in the microfluidic chip for simultaneous determina-tion of interferon-g [24], an integrated 2-D microfluidic beads array for single-base detection [25] and cardiac risk assessment [26], and the microfluidic-based microarray for DNA hybridization analysis [27][28][29][30]. Beads assay technologies have become more important for their remarkable advantages in comparison with the spotted array technology [31][32][33], and their use has expanded into many areas of medicine and biology, such as fiber optic beads array for SNP analysis [34], cytometric beads array for species identification of microbiology [35], a planar beads array for gene expression analysis [36], etc. Recently, we reported a novel 1-D microfluidic beads array for rapid and quantitative molecular profiling by using a two-site antibody "sandwich" format, which integrated rapid binding kinetics and multiplex-encoding capabilities of suspension bead carriers and the liquid handling advantages of microfluidic devices [37].…”

Section: Introductionmentioning

confidence: 99%

Detection of single‐base mutations using 1‐D microfluidic beads array

et al. 2007

View full text Add to dashboard Cite

The application of a 1-D microfluidic beads array that is composed of individually addressable functionalized SiO2 beads has been demonstrated for detection of single-base mutations based on "sandwich" hybridization assay without additional sample labeling and PCR amplification. We concentrated on detection of mutations in the human p53 tumor suppressor gene with more than 50% mutation frequency in the known human cancers. Using a microinjection system, functionalized beads could be selectively and linearly arrayed in a single microfluidic channel comprising many periodic chambers. This 1-D microfluidic beads array was sufficiently sensitive to identify single-nucleotide mutations in 40 pM quantities of DNA targets and could discriminate the mutated alleles in an excess of nonmutated alleles at a level of one mutant in 100 wild-type sequences. The surface of beads was regenerated and rehybridized up to six times without obvious loss of signal. The entire reaction process was done at room temperature within minutes, and only 2-10 microL sample solution was needed to complete the whole detection process. The p53 genotypes of A549, CNE2, and SKBr-3 cell lines were also correctly evaluated by using mRNA extracts as target without need for sample labeling and amplification. Thus, this platform enabled rapid and exact discrimination of gene mutations with the advantages of reusability, simple handling of liquid, low cost, and little reagent consumption.

show abstract

“… 24 , 25 Recently, the advancement of molecular diagnostics allows an innovative agglutination format through nucleic acid hybridization to detect amplicons of polymerase chain reaction (PCR) or other nucleic acid targets. 22 , 26 − 28 We harness the molecular approach that immobilizes a pair of oligonucleotide probes on microparticles, respectively, followed by agglutination formation against target bacterial nucleic acid through a hybridization reaction. Because there are more than 10 000 copies of 16S ribosomal ribonucleic acid (rRNA) per bacterial cell, 29 this high abundance not only allows us to circumvent drawbacks of nucleic acid amplification, but also ensures high sensitivity against target bacteria.…”

mentioning

confidence: 99%

“…In this paper, we propose a low-cost microfluidic imaging flow cytometry platform for rapid UTI diagnostics. The platform utilizes the narrow beam scanning (NBS) technique with off-the-shelf complementary metal-oxide-semiconductor (CMOS) imagers to collect images from molecular agglutination bioassays. , The agglutination assay is a simple approach to produce signals detected by various biosensors. − These bioassays have mostly been demonstrated by means of antibody-coated microparticles for detection of biomarkers or bacteria. , Recently, the advancement of molecular diagnostics allows an innovative agglutination format through nucleic acid hybridization to detect amplicons of polymerase chain reaction (PCR) or other nucleic acid targets. ,− We harness the molecular approach that immobilizes a pair of oligonucleotide probes on microparticles, respectively, followed by agglutination formation against target bacterial nucleic acid through a hybridization reaction. Because there are more than 10 000 copies of 16S ribosomal ribonucleic acid (rRNA) per bacterial cell, this high abundance not only allows us to circumvent drawbacks of nucleic acid amplification, but also ensures high sensitivity against target bacteria.…”

mentioning

confidence: 99%

A Rapid and Low-Cost Pathogen Detection Platform by Using a Molecular Agglutination Assay

Chen

Wang

et al. 2018

ACS Cent. Sci.

View full text Add to dashboard Cite

Rapid and low-cost pathogen diagnostic approaches are critical for clinical decision-making procedures. Cultivating bacteria often takes days to identify pathogens and provide antimicrobial susceptibilities. The delay in diagnosis may result in compromised treatment and inappropriate antibiotic use. Over the past decades, molecular-based techniques have significantly shortened pathogen identification turnaround time with high accuracy. However, these assays often use complex fluorescent labeling and nucleic acid amplification processes, which limit their use in resource-limited settings. In this work, we demonstrate a wash-free molecular agglutination assay with a straightforward mixing and incubation step that significantly simplifies procedures of molecular testing. By targeting the 16S rRNA gene of pathogens, we perform a rapid pathogen identification within 30 min on a dark-field imaging microfluidic cytometry platform. The dark-field images with low background noise can be obtained using a narrow beam scanning technique with off-the-shelf complementary metal oxide semiconductor (CMOS) imagers such as smartphone cameras. We utilize a machine learning algorithm to deconvolute topological features of agglutinated clusters and thus quantify the abundance of bacteria. Consequently, we unambiguously distinguish Escherichia coli positive from other E. coli negative among 50 clinical urinary tract infection samples with 96% sensitivity and 100% specificity. Furthermore, we also apply this quantitative detection approach to achieve rapid antimicrobial susceptibility testing within 3 h. This work exhibits easy-to-use protocols, high sensitivity, and short turnaround time for point-of-care testing uses.

show abstract

A simple nucleic acid hybridization/latex agglutination assay for the rapid detection of polymerase chain reaction amplicons

Cited by 4 publications

References 41 publications

Nanoslit membrane-integrated fluidic chip for protein detection based on size-dependent particle trapping

Nanoslit membrane-integrated fluidic chip for protein detection based on size-dependent particle trapping

Detection of single‐base mutations using 1‐D microfluidic beads array

A Rapid and Low-Cost Pathogen Detection Platform by Using a Molecular Agglutination Assay

Contact Info

Product

Resources

About