Multiplex optical detection and quantification of DNA fragments by metallo-peptide assemblies.
Ontology highlight
ABSTRACT:
SUBMITTER: Saha A
PROVIDER: S-EPMC6584589 | biostudies-literature | 2019 Jun
REPOSITORIES: biostudies-literature
Publications
Multiplex optical detection and quantification of DNA fragments by metallo-peptide assemblies.
Saha Abhijit A Reches Meital M
Scientific reports 20190619 1
Rapid detection of infectious agents such as bacteria and viruses are important for proper health management, agriculture and homeland security. This paper presents a multiplex DNA detection system self-assembled by a metallo-peptide complex. Within five minutes, the system can simultaneously detect multiple DNA fragments, without any need for their separation. The presence of proteins in the sample does not harm the detection capabilities of the system, which can discriminate even between one b ...[more]
Similar Datasets
Project description:We describe PieceMaker, a software tool for the design of applications of selector probes-oligonucleotide probes that direct circularization of target nucleic acid molecules. Such probes can be combined in parallel to circularize a selection of fragments from restriction digested total genomic DNA. These fragments can then be amplified in a single PCR using a common primer pair, yielding substrates for subsequent analyses, such as parallel genotyping or sequencing. However, designing multiplex selector assays is a laborious task. The PieceMaker program alleviates this problem by selecting restriction enzymes to generate suitable fragments for selection, and generating the output data required to design the selector probes.
| S-EPMC1087790 | biostudies-literature
Project description:Efficient treatments in bacterial infections require the fast and accurate recognition of pathogens, with concentrations as low as one per milliliter in the case of septicemia. Detecting and quantifying bacteria in such low concentrations is challenging and typically demands cultures of large samples of blood (~1 milliliter) extending over 24-72 hours. This delay seriously compromises the health of patients. Here we demonstrate a fast microorganism optical detection system for the exhaustive identification and quantification of pathogens in volumes of biofluids with clinical relevance (~1 milliliter) in minutes. We drive each type of bacteria to accumulate antibody functionalized SERS-labelled silver nanoparticles. Particle aggregation on the bacteria membranes renders dense arrays of inter-particle gaps in which the Raman signal is exponentially amplified by several orders of magnitude relative to the dispersed particles. This enables a multiplex identification of the microorganisms through the molecule-specific spectral fingerprints.
| S-EPMC4929498 | biostudies-literature
Project description:We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs. Important qPCR experimental details and information that is critical to performance and reliability of assay results were investigated. Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods. To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction. As the reaction volume decreased, qPCR assays became more efficient with 1 µl reaction master mix being the most efficient. For more accurate quantification of parasites in a sample, we developed plasmid DNAs for all the three assay targets for absolute quantification. All of absolute qPCR assays performed with efficiency of more than 94%, R(2) values greater than 0.99 and the STDEV of each replicate was <0.167. Linear regression plots generated from absolute qPCR assays were used to estimate the corresponding parasite density from relative qPCR in terms of parasite/µl. One copy of plasmid DNA was established to be equivalent to 0.1 parasite/µl for Plasmodium spp. assay, 0.281 parasites for P. falciparum assay and 0.127 parasite/µl for P. vivax assay. This study demonstrates for the first time use of plasmid DNA in absolute quantification of malaria parasite. The use of plasmid DNA standard in quantification of malaria parasite will be critical as efforts are underway to harmonize molecular assays used in diagnosis of malaria.
| S-EPMC3756973 | biostudies-literature
Project description:Nanoscale plasmonic assemblies display exceptionally strong chiral optical activity. So far, their structural design was primarily driven by challenges related to metamaterials whose practical applications are remote. Here we demonstrate that gold nanorods assembled by the polymerase chain reaction into DNA-bridged chiral systems have promising analytical applications. The chiroplasmonic activity of side-by-side assembled patterns is attributed to a 7-9 degree twist between the nanorod axes. This results in a strong polarization rotation that matches theoretical expectations. The amplitude of the bisignate 'wave' in the circular dichroism spectra of side-by-side assemblies demonstrates excellent linearity with the amount of target DNA. The limit of detection for DNA using side-by-side assemblies is as low as 3.7 aM. This chiroplasmonic method may be particularly useful for biological analytes larger than 2-5 nm which are difficult to detect by methods based on plasmon coupling and 'hot spots'. Circular polarization increases for inter-nanorod gaps between 2 and 20 nm when plasmonic coupling rapidly decreases. Reaching the attomolar limit of detection for simple and reliable bioanalysis of oligonucleotides may have a crucial role in DNA biomarker detection for early diagnostics of different diseases, forensics and environmental monitoring.
| S-EPMC3826651 | biostudies-literature
Project description:Homogenous protein assays, despite the potential for mix-and-read workflows, have eluded widespread acceptance due to interferences in biological matrices and limited multiplexability. Here, we employ standard qPCR instrumentation for thermofluorimetric analysis of bivalent probe (TFAB) assemblies, allowing protein levels to be quantitatively translated into multiplexable DNA melting transitions within 30 min. As protein-bound bivalent probes are thermodynamically more stable than unbound probes, differential thermal analysis can remove background analytically, without physical separation. Using either antibody-oligonucleotides or aptamers as probes, TFAB is validated for protein quantification in buffer, human serum, and human plasma and for assaying hormone secretions from endocrine cells. The direct optical method exhibits superior scalability, allowing detection of only 1 amol of protein in microfluidic channels of 100 pL volume. Overall, we demonstrate TFAB as a robust and generalizable homogeneous protein assay with superior performance in biological matrices.
| S-EPMC4601099 | biostudies-literature
Project description:We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.
| S-EPMC1087789 | biostudies-literature
Project description:BackgroundMultiplex PCR has been successfully applied in many areas since it was first reported in 1988; however, it suffers from poor universality.ResultsA novel method called Universal Multiplex PCR (UM-PCR) was created, which simultaneously amplifies multiple target fragments from genomic DNA. The method has two steps. First, the universal adapter-F and universal adapter-R are connected to the forward primers and the reverse primers, respectively. Hairpin structures and cross dimers of five pairs of adapter-primers are detected. Second, UM-PCR amplification is implemented using a novel PCR procedure termed "Two Rounds Mode" (three and 28-32 cycles). The first round (the first three cycles) is named the "One by One Annealing Round". The second round (28-32 cycles) combines annealing with extension. In the first two cycles of the first round, primers only amplify the specific templates; there are no templates for the universal adapters. The templates of universal adapters begin to be synthesized from the second cycle of the first round, and universal adapters and primers commence full amplification from the third cycle of the first round.ConclusionsUM-PCR greatly improves the universality of multiplex PCR. UM-PCR could rapidly detect the genetic purity of maize seeds. In addition, it could be applied in other areas, such as analysis of polymorphisms, quantitative assays and identifications of species.
| S-EPMC3485162 | biostudies-literature
Project description:The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.
| S-EPMC5064307 | biostudies-literature
Project description:Elevated levels of transfer RNA (tRNA) fragments were recently identified in plasma samples from people with epilepsy in advance of a seizure, indicting a potential novel class of circulating biomarker. Current methods for detection and quantitation of tRNA fragments (tRFs) include northern blotting, RNA sequencing or custom Taqman-based PCR assays. The development of a simple, at home or clinic-based test, would benefit from a simple and reliable method to detect the tRFs using small volumes of biofluids. Here we describe an electrochemical direct detection method based on electrocatalytic platinum nanoparticles to detect 3 specific tRFs: 5'AlaTGC, 5'GlyGCC, and 5'GluCTC. Using synthetic tRF mimics we showed this system was linear over 9 orders of magnitude with sub-attomolar limits of detection. Specificity was tested using naturally occurring mismatched tRF mimics. Finally, we quantified tRF levels in patient plasma and showed that our detection system recapitulates results obtained by qPCR. We have designed a tRF detection system with high sensitivity and specificity capable of quantifying tRFs in low volumes of plasma using benchtop apparatus. This is an important step in the development of a point-of-care device for quantifying tRFs in whole blood.
| S-EPMC7200677 | biostudies-literature
Project description:The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C-terminal fragments of the protein TDP-43. Here, we tested the hypothesis that highly sensitive mass spectrometry with parallel reaction monitoring (MS-PRM) can generate a high-resolution map of pathological TDP-43 peptide ratios to form the basis for quantitation of abnormal C-terminal TDP-43 fragment enrichment. Human cortex and spinal cord, microscopically staged for the presence of p-TDP-43, p-tau, alpha-synuclein, and beta-amyloid pathology, were biochemically fractionated and analyzed by immunoblot and MS for the detection of full-length and truncated (disease-specific) TDP-43 peptides. This informed the synthesis of heavy isotope-labeled peptides for absolute quantification of TDP-43 by MS-PRM across 16 ALS, 8 Parkinson's, 8 Alzheimer's disease, and 8 aged control cases. We confirmed by immunoblot the previously described enrichment of pathological C-terminal fragments in ALS-TDP urea fractions. Subsequent MS analysis resolved specific TDP-43 N- and C-terminal peptides, including a novel N-terminal truncation site-specific peptide. Absolute quantification of peptides by MS-PRM showed an increased C:N-terminal TDP-43 peptide ratio in ALS-TDP brain compared to normal and disease controls. A C:N-terminal ratio >1.5 discriminated ALS from controls with a sensitivity of 100% (CI 79.6-100) and specificity of 100% (CI 68-100), and from Parkinson's and Alzheimer's disease with a sensitivity of 93% (CI 70-100) and specificity of 100% (CI 68-100). N-terminal truncation site-specific peptides were increased in ALS in line with C-terminal fragment enrichment, but were also found in a proportion of Alzheimer cases with normal C:N-terminal ratio but coexistent limbic TDP-43 neuropathological changes. In conclusion this is a novel, sensitive, and specific method to quantify the enrichment of pathological TDP-43 fragments in human brain, which could form the basis for an antibody-free assay. Our methodology has the potential to help clarify if specific pathological TDP-43 peptide signatures are associated with primary or secondary TDP-43 proteinopathies.
| S-EPMC8412074 | biostudies-literature