Sandwich DNA Hybridization Research Articles

Flow Cytometric Detection of Bacillus spoOA Gene in Biofilm Using Quantum Dot Labeling

Quantum dot (QD)-induced fluorescence detection of bead-based DNA sandwich hybridization was studied for rapid analysis of the Bacillus spoOA gene in biofilms. Hybridization between two DNA probes and target DNA occurred and the hybridization signal was detected in a flow cytometer. To prepare the bead-DNA capture and QD-DNA detection probes, the coupling or bioconjugation reactions were carefully controlled. It was successfully demonstrated that the fluorescence response of the hybrid complex was linear in the range of 3.2-1000 nM of synthetic target DNA (R(2) = 0.97) and that the detection limit was 0.02 nM. An optimized labeling method and bead-based DNA hybridization were then applied to real PCR products from a biofilm sample with satisfactory results (R(2) = 0.94), thereby confirming that the proposed assay provides a rapid, sensitive, and specific method for Bacillus spoOA gene detection in bofilms. This approach enables multiple target detection using multicolor QD-DNA probes in a shorter time.

Optical Detection of Human Papillomavirus Type 16 and Type 18 by Sequence Sandwich Hybridization With Oligonucleotide-Functionalized Au Nanoparticles

The importance of detecting and subtyping human papillomaviruses (HPVs) in clinical and epidemiological studies has been well addressed. In detecting the most common types of HPV, type 16 (HPV-16) and type 18 (HPV-18), in the cervical mucous of patients in a simple and rapid manner, the assay of a label-free colorimetric DNA sensing method based on sequence sandwich hybridization with oligonucleotide-functionalized Au nanoparticles (AuNPs) was fabricated in this study. Specific oligonucleotide probes were designed for the sequence detection within the L1 gene of HPV-16 and HPV-18, and the probes were capped onto AuNPs, as AuNP probes. The target HPV sequences in clinical specimens were obtained by an asymmetric polymerase chain reaction (PCR) with universal primers, which can amplify the target sequences from several HPV serotypes, including HPV-16 and HPV-18. The DNA sandwich hybridization between the target sequences and the specific AuNP probes was performed at a temperature closer to the theoretical melting temperature of the DNA hybridization. Next, the procedure of increasing salt concentration and cooling the hybridizing solution was immediately utilized to discriminate the target sequences of HPV-16 or HPV-18. If the target sequences were not complementary to sequences of AuNP probes, the AuNPs would aggregate because no duplex DNA formation occurred such that the color of the reaction solution changed from red to purple. If the AuNP probes were a perfect match to the target sequences and a full DNA sandwich hybridization occurred, the reaction solution maintained its red color. A total of 70 mucous specimens from patients with cervical intraepithelial neoplasia were tested by the AuNP probes sandwich hybridization. The results show that there were 33, 16, 5, and 16 cases detected with HPV-16, HPV-18, both HPV-16 and HPV-18 (HPV-16/HPV-18), and neither HPV-16 nor HPV-18, respectively.

Magnetic bead-based DNA hybridization assay with chemiluminescence and chemiluminescent imaging detection

Simple and sensitive chemiluminescence (CL) and CL imaging methods have been developed for the magnetic bead-based DNA hybridization assay. The assay relies on the high sensitivity and long stable light signal of the CL system in which horseradish peroxidase (HRP) catalyzes the luminol-H(2)O(2) reaction with para-iodophenol (PIP) as the enhancer. In this protocol, a sandwich DNA hybridization is performed by mixing the target DNA with the magnetic bead-captured DNA and the biotinylated reporter DNA, followed through the biotin-streptavidin reaction with conjugated HRP, and then the conjugated HRP is determined by the CL system. The proposed CL protocol is suitable for for the detection of sequence-specific DNA related to the avian influenza A H1N1 virus at levels as low as 10 amol, and the CL imaging detection has a similar sensitivity. The sensitivities of the proposed methods with the HRP label are better than most of the metal nanoparticle-based methods, and are comparable with that of utilizing amplified techniques for DNA hybridization detection. In addition, the perfectly complementary DNA sequences and the single-base mismatched DNA sequences can be better distinguished by a thermally-stringent hybridization and washing steps. So, the proposed CL method can offer great promise for single-nucleotide polymorphism (SNP) analysis. Moreover, the proposed method may have significant potential for the simultaneous detection of various DNA sequences when different capture DNA sequences and reporter DNA sequences are used in a microarray.

Electrochemical Imaging of Localized Sandwich DNA Hybridization Using Scanning Electrochemical Microscopy

Imaging of localized hybridization of nucleic acids immobilized on gold-DNA chip was performed by means of the feedback mode of scanning electrochemical microscopy (SECM). Thiol-tethered oligodeoxynucleotide (HS-ODN) probes, spotted on a gold surface, were hybridized with unmodified target sequence via sandwich hybridization with a biotinylated signaling probe. Spots where sequence-specific hybridization had occurred were developed by adding a streptavidin-alkaline phosphatase conjugate and biocatalyzed precipitation of an insoluble and insulating product. As a consequence, the surface conductivity of the spotted region of the chip where hybridization had taken place changed. These changes in conductivity were sensitively detected by the SECM tip. The proposed method allows imaging of a DNA array in a straightforward way. Analysis of real samples was also performed coupling this method with polymerase chain reaction. The imaging of 60 nM PCR amplicon (255 bp) was demonstrated.

Sensitized Luminescent Terbium Nanoparticles: Preparation and Time-Resolved Fluorescence Assay for DNA

A highly luminescent terbium nanoparticle as the biolabel based on the sensitization of a dye molecule was prepared. The luminescent complexes included in the particles were composed of a quinolone-based dye molecule as the light-energy transfer donor and a polyaminocarboxylate-based chelator with excellent water-solubility and a high binding constant for lanthanides. The structure of two functional entities in the single molecule made the complex highly luminescent in aqueous solution. Silica nanoparticles containing terbium complexes were prepared by the reverse microemulsion method. Such a terbium nanoparticle is as bright as about 340 free terbium complexes, and it has a 1.5-ms fluorescence lifetime that enables it to be used in the time-resolved fluorescence assays. The conjugate of the nanoparticle with oligonucleotide was prepared and used to carry out a DNA sandwich hybridization assay based on magnetic microbeads as solid-phase carrier. The experimental results showed that the detection sensitivity with the nanoparticles is more than 100-fold as high as that with dye Fluorescein isothiocyanate (FITC) molecules.

Oligonucleotide synthesis onto poly(N‐acryloylmorpholine‐co‐N‐acryloxysuccinimide): Assessment of the resulting conjugates in a DNA sandwich hybridization test

AbstractA water‐soluble statistical poly(N‐acryloylmorpholine‐co‐N‐acryloxysuccinimide) [poly(NAM/NAS)] copolymer was studied for polymer–oligonucleotide (ODN) conjugate elaboration and for further use in diagnostic applications. Three different copolymers were first prepared by free‐radical solution polymerization with different N‐acryloylmorpholine (NAM) and N‐acryloxysuccinimide (NAS) molar ratios (80/20, 70/30, and 60/40). Their number‐average molecular weights ranged from 98,000 to 120,000 g/mol, as determined by aqueous size exclusion chromatography with an online light‐scattering detector. Then, polymer–ODN conjugates were obtained via a strategy consisting of the direct synthesis of ODNs onto polymer chains previously grafted onto a controlled pore glass support. Before the grafting of the polymer onto the solid support, a preliminary step was performed to bind a nucleotide starter along the polymer chain (via the reactive NAS units) to initiate automated DNA synthesis. To multiply the number of ODNs growing from starters, a branched phosphoramidite synthon [bearing two O‐dimethoxytrityl groups] was introduced at the first step of ODN elongation as a short sequence of four branched synthons alternated with three thymidine residues. Conjugates were assessed in a DNA sandwich hybridization test developed for hepatitis B virus detection. Sensitivity limits were evaluated and compared to those obtained with an other polymer, poly(maleic anhydride‐alt‐methyl vinyl ether) [poly(MA/MVE)]. A sensitivity limit of 2.6 × 107 DNA copies/mL was reached with the poly(MA/MVE)–ODN conjugate at the capture phase and with the poly(NAM/NAS)–branched ODN conjugate at the detection phase of the test. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 3784–3795, 2004

Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLink™ NH microwell plate sandwich hybridization

Outbreak of diseases associated with consumption of raw shellfish especially oysters is a major concern to the seafood industry and public health agencies. A multiplex PCR amplification of targeted gene segments followed by DNA–DNA sandwich hybridization was optimized to detect the etiologic agents. First, a multiplex PCR amplification of hns, spvB, vvh, ctx and tl was developed enabling simultaneous detection of total Salmonella enterica serotype Typhimurium, Vibrio vulnificus, Vibrio cholerae and Vibrio parahaemolyticus from both pure cultures and seeded oysters. Amplicons were then subjected to a colorimetric CovaLink™ NH microwell plate sandwich hybridization using phophorylated and biotinlylated oligonucleotide probes, the nucleotide sequences of which were located internal to the amplified DNA. The results from the hybridization with the multiplexed PCR amplified DNA exhibited a high signal/noise ratio ranging between 14.1 and 43.2 measured at 405 nm wavelength. The sensitivity of detection for each pathogen was 10 2 cells/g of oyster tissue homogenate. The results from this study showed that the combination of the multiplex PCR with a colorimetric microwell plate sandwich hybridization assay permits a specific, sensitive, and reproducible system for the detection of the microbial pathogens in shellfish, thereby improving the microbiological safety of shellfish to consumers.

An electrochemical detection scheme for identification of single nucleotide polymorphisms using hairpin-forming probes.

Single nucleotide polymorphisms are implicated as having a significant role in regulating growth, development and, thereby, human health and disease. We have developed a method for identifying single nucleotide genetic alterations by combining hairpin-forming DNA probes and electrochemical detection of sandwich DNA hybridization. Incorporation of hairpin-forming competitor probes and the catalyzed reporter deposition amplification system further improves assay specificity by 7-fold and sensitivity by 100-fold. We have demonstrated that the system successfully identified the factor V Leiden mutations from human blood specimens.

Rapid colorimetric hybridization assay for detecting amplified Helicobacter pylori DNA in gastric biopsy specimens.

A very simple, practical, sensitive, and specific colorimetric hybridization assay for detecting amplified Helicobacter pylori DNA is described. This assay, which combines a sensitive sandwich DNA hybridization reaction and a colorimetric protocol similar to those used in conventional enzyme immunoassays, was shown to be suitable for detecting H. pylori-infected gastric biopsy specimens and for monitoring the eradication of the pathogen after treatment. The specificity and sensitivity of the colorimetric hybridization assay were tested by assaying 27 H. pylori strains (4 reference and 23 clinical isolates), 9 strains of other Helicobacter spp. or Campylobacter spp., and 11 clinical isolates of other urease-positive bacteria. The likelihood of H. pylori detection in gastric biopsy specimens by the colorimetric hybridization assay was evaluated with 23 H. pylori-positive and 41 H. pylori-negative biopsy specimens on the basis of positive and negative results, respectively, of culture, rapid urease test, histological examination, and PCR. Biopsy specimens from 33 treated patients, endoscopied 4 to 8 weeks after the end of treatment, were also tested. All H. pylori strains showed positive results in the colorimetric hybridization assay, presenting optical densities at 450 nm (OD450S) of > or = 3.0. None of the other Helicobacter spp., Campylobacter spp., or the clinical isolates of other urease-positive bacteria showed OD450S equal to or greater than the cutoff (mean OD450 cutoff, 0.208). The colorimetric hybridization assay detected all 23 H. pylori-positive biopsy specimens (mean OD450, 2.910 +/- 0.295), while none of the H. pylori-negative biopsy specimens was shown to be positive in the assay (mean OD450, 0.108 +/- 0.025). H. pylori was considered to be not eradicated from three of the posttreatment biopsy specimens by culture, rapid urease test, histological examination, and PCR. They were all positive by the colorimetric hybridization assay, and their OD450S were > or = 3.0. The colorimetric hybridization assay also detected two other H. pylori-positive patients. Specimens from these two patients had negative culture, rapid urease test, and histology results, and a specimen from one of them also tested negative by PCR. These results indicate that the colorimetric hybridization assay is a suitable method both for the diagnosis of H. pylori in biopsy specimens and for the follow-up of patients after the end of treatment.

A colorimetric microtiter plate sandwich DNA hybridization system for the detection of Salmonella in foods

A microtiter plate sandwich DNA hybridization system for the specific detection of Salmonella-contaminated food samples was developed. The capture probe of this system was a single strand plasmid M13 DNA with a 0.4 kb HindIII- EcoRI insert, while the detection probe was a 1.4 kb HindIII- EcoRI DNA fragment prepared from the replication form (RF) of a recombinant M13 DNA containing this 1.4 kb fragment. Labeling of the detection probe was performed by nick-translation using biotin-21-dUTP. Both the 0.4 kb and the 1.4 kb HindIII- EcoRI DNA fragments were derived from a 1.8 kb HindIII DNA fragment which under specific conditions could hybridize with all the Salmonella isolates tested but not with any of the non- Salmonella strains including Enterobacteriaceae, a species which is closely related to Salmonella. When the capture probe used for microtiter plate coating was 2 μg per well while the detection probe was 100 ng per well, target DNA obtained from 10 3 cells of Salmonella could be detected without ambiguity. When this system was used for the detection of Salmonella artificially added to food samples such as milk, pork and beef, the results showed that this system was reliable, although target DNA prepared from one ml of the mixture containing N × 10 6 cells was required for each microtiter well to yield a positive reaction. Examination of the storage stability of the microtiter plates precoated with capture probes showed that within 16 d of storage at 8°C, these microtiter plates were reliable for the detection of Salmonella.

OmpA gene in the detection of Escherichia coli and other Enterobacteriaceae by nucleic acid sandwich hybridization.

The applicability of the DNA sandwich hybridization method to detection of bacterial DNA from crude samples is demonstrated using Escherichia coli as a model. In sandwich hybridization the sample DNA mediates the binding of a labeled probe DNA fragment to a second DNA bound on filter. For this study the DNA reagents were prepared by subclonings from a recombinant plasmid containing the E. coli K-12 ompA gene and an adjacent fragment of E. coli DNA. The 5' half of the ompA gene (738 base pairs) in pBR322 served as the filter reagent. The 3' half of the ompA gene (300 base pairs) and the adjacent 1,500 base pairs of E. coli DNA were cloned into the single-stranded phage vector M13mp7, and the resulting recombinant phage DNA was labeled with 125I and used as probe in the hybridizations. For maximal hybridization the DNA reagents had to be present in excess of the sample nucleic acid, which was preferably fragmented before testing. In the optimized test, 3 X 10(6) molecules of E. coli DNA from lysed cells were detected by an overnight reaction; the sensitivity of the test was not affected by the presence of 10(9) unrelated bacteria. With the ompA reagents, all members of the family Enterobacteriaceae tested were detected even if the sensitivity was decreased as compared with that for the homologous bacteria. With all other bacteria, including aerobic and anaerobic species of clinical importance, the test was negative. These findings suggest that it may be possible to find group-specific reagents to be used in diagnostic bacteriology.