Rapid and reliable detection of 11 food-borne pathogens using thin-film biosensor chips

Traditional methods for identifying food-borne pathogens are time-consuming and laborious, so it is necessary to develop innovative methods for the rapid identification of food-borne pathogens. Here, we report the development of silicon-based optical thin-film biosensor chips for sensitive detection of 11 food-borne pathogens. Briefly, aldehyde-labeled probes were arrayed and covalently attached to a hydrazine-derivatized chip surface, and then, biotinylated polymerase chain reaction (PCR) amplicons were hybridized with the probes. After washing and brief incubation with an antibiotin immunoglobulin G–horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate, biotinylated chains bound to the probes were visualized as a color change on the chip surface (gold to blue/purple). Highly sensitive and accurate examination of PCR fragment targets can be completed within 30 min. This assay is extremely robust, sensitive, specific, and economical and can be adapted to different throughputs. Thus, a rapid, sensitive, and reliable technique for detecting 11 food-borne pathogens was successfully developed.     

Use of a thin film biosensor for rapid visual detection of PCR products in a multiplex format

Rapid, sensitive assays for nucleic acid amplification products have utility for the identification of bacterial or viral infections. We have developed a nucleic acid hybridization assay utilizing thin film technology that permits visual detection of hybrids. The silicon-based biosensor detects the presence of target sequences by enzymatically transducing the formation of nucleic acid hybrids into molecular thin films. These films alter the interference pattern of light on the biosensor surface, producing a perceived color change. We have applied this technology to the development of a chip containing capture probes specific for human respiratory virus sequences including respiratory syncytial virus, influenza virus A and B, parainfluenza virus types 1 and 3, and rhinovirus. In a ten-minute assay, the biosensor permits unambiguous identification of viral-specific RT/PCR products from infected cell lysates.     

Single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor microchips

We have developed a rapid assay for single nucleotide polymorphism (SNP) detection that utilizes electronic circuitry on silicon microchips. The method was validated by the accurate discrimination of blinded DNA samples for the complex quadra-allelic SNP of mannose binding protein. The microchip directed the transport, concentration, and attachment of amplified patient DNA to selected electrodes (test sites) creating an array of DNA samples. Through control of the electric field, the microchip enabled accurate genetic identification of these samples using fluorescently labeled DNA reporter probes. The accuracy of this approach was established by internal controls of dual labeled reporters and by using mismatched sequences in addition to the wild-type and variant reporter sequences to validate the SNP-genotype. The ability to customize this assay for multiple genes has advantages over other existing approaches.     

A New Restriction Endonuclease-Based Method for Highly-Specific Detection of DNA Targets from Methicillin-Resistant Staphylococcus aureus

PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP). At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i) any mutation of the restriction site reduced the signal to zero; (ii) double and triple point mutations of sequences flanking the restriction site reduced restriction to 50–80% of the positive control; and (iii) a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same unpurified mixture in broad-range PCR applications.     

Simultaneous detection of dual single-base mutations by capillary electrophoresis using quantum dot-molecular beacon probe

Here a novel capillary electrophoresis (CE) for simultaneous detection of dual single-base mutations using quantum dot-molecular beacon (QD-MB) probe is described. Two QD-MB probes were designed using 585 and 650-nm emitting CdTe QDs which were covalently conjugated to MBs with different DNA oligonucleotide sequences by amide linkage and streptavidin-biotin binding, respectively. The hybridizations of QD-MB probes with different DNA targets were then monitored by CE, and results indicated that the two QD-MB probes specifically hybridized with their complementary DNA sequences, respectively. Target DNA identification was observed to have a high sensitivity of 16.2 pg in CE. Furthermore, the simultaneous detection of dual single-base mutations in a given DNA oligonucleotide was successfully achieved in CE using above two QD-MB probes. This novel CE-assisted QD-MB biosensor offers a promising approach for simultaneous detection of multiple single-base mutations, and exhibits potential capability in the single nucleotide polymorphism (SNP) analysis and high-sensitivity DNA detection.     

Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy

We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5′ end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10⁻⁸ M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10⁻¹¹ M solutions of PCR amplicons from M.tuberculosis in only 100 s.     

Comparative studies on species identification of Noctuoidea moths in two nature reserve conservation zones (Beijing,China) using DNA barcodes and thin‐film biosensor chips

Rapid and accurate identification of species is required for the biological control of pest Noctuoidea moths. DNA barcodes and thin‐film biosensor chips are two molecular approaches that have gained wide attention. Here, we compare these two methods for the identification of a limited number of Noctuoidea moth species. Based on the commonly used mitochondrial gene cytochrome c oxidase I (the standard DNA barcode for animal species), 14 probes were designed and synthesized for 14 species shared by two national nature reserves in Beijing and Hebei, China. Probes ranged in length from 18 to 27 bp and were designed as mismatch probes to guarantee that there were at least three base differences between the probe and nontarget sequences. The results on the chip could be detected by the naked eye without needing special equipment. No cross‐hybridizations were detected although we tested all probes on the 14 target and 24 nontarget Noctuoidea species. The neighbour‐joining tree of the 38 species based on COI sequences gave 38 highly supported independent groups. Both DNA barcoding and thin‐film biosensor chips, based on the COI gene, are able to accurately identify and discriminate the 14 targeted moth species in this study. Because of its speed, high accuracy and low cost, the thin‐film biosensor chip is a very practical means of species identification. Now, a more comprehensive chip will be developed for the identification of additional Noctuoidea moths for pest control and ecological protection.     

Electrochemically amplified molecular beacon biosensor for ultrasensitive DNA sequence-specific detection of Legionella sp

An electrochemically amplified molecular beacon (EAMB) biosensor is constructed using thiolated hairpin DNA-ferrocene probes on gold electrode. The switching from "on" to "off" states of individual probes in the presence of complementary DNA target influences the electrode potential, besides the current, owing to changes in surface density of the electroactive hairpin DNA-ferrocene probes. The EAMB biosensor demonstrates linear range over 8 orders of magnitude with ultrasensitive detection limit of 2.3 × 10(-14)M for the quantification of a 21-mer DNA sequence. Its applicability is tested against PCR amplicons derived from genomic DNA of live Legionella pneumophila. Excellent specificity down to one and three nucleotides mismatches in another strain of L. pneumophila and a different bacterium species, respectively, is demonstrated.     

DNA-probes for the highly sensitive identification of single nucleotide polymorphism using single-molecule spectroscopy

This article presents a new, highly sensitive method for the identification of single nucleotide polymorphisms (SNPs) in homogeneous solutions using fluorescently labeled hairpin-structured oligonucleotides (smart probes) and fluorescence single-molecule spectroscopy. While the hairpin probe is closed, fluorescence intensity is quenched due to close contact between the chromophore and several guanosine residues. Upon hybridization to the respective target SNP sequence, contact is lost and the fluorescence intensity increases significantly. High specificity is achieved by blocking sequences containing mismatch with unlabeled oligonucleotides. Time-resolved single-molecule fluorescence spectroscopy enables the detection of individual smart probes passing a small detection volume. This method leads to a subnanomolar sensitivity for this single nucleotide specific DNA assay technique.     

Guidelines for incorporating non-perfectly matched oligonucleotides into target-specific hybridization probes for a DNA microarray

Sequence-specific oligonucleotide probes play a crucial role in hybridization techniques including PCR, DNA microarray and RNA interference. Once the entire genome becomes the search space for target genes/genomic sequences, however, cross-hybridization to non-target sequences becomes a problem. Large gene families with significant similarity among family members, such as the P450s, are particularly problematic. Additionally, accurate single nucleotide polymorphism (SNP) detection depends on probes that can distinguish between nearly identical sequences. Conventional oligonucleotide probes that are perfectly matched to target genes/genomic sequences are often unsuitable in such cases. Carefully designed mismatches can be used to decrease cross-hybridization potential, but implementing all possible mismatch probes is impractical. Our study provides guidelines for designing non-perfectly matched DNA probes to target DNA sequences as desired throughout the genome. These guidelines are based on the analysis of hybridization data between perfectly matched and non-perfectly matched DNA sequences (single-point or double-point mutated) calculated in silico. Large changes in hybridization temperature predicted by these guidelines for non-matched oligonucleotides fit independent experimental data very well. Applying the guidelines to find oligonucleotide microarray probes for P450 genes, we confirmed the ability of our point mutation method to differentiate the individual genes in terms of thermodynamic calculations of hybridization and sequence similarity.     

用反向斑点杂交方法检测猪常见致病菌

目的:建立检测猪常见致病菌的反向斑点杂交方法。方法:将23S rRNA基因芯片用的针对12种细菌的25～30 mer探针加长到30～38 mer,2对通用引物序列不变。用地高辛标记下游引物,以尼龙膜为载体制备膜芯片,检验探针/膜杂交的特异性和敏感性;另外设计1条大肠杆菌K88基因探针、一段带K88探针的报告基因和1对报告基因的反向PCR引物,在PCR体系中增加封口的K88报告基因和反向引物对,被检样品扩增后进行膜杂交。结果:修改的13条探针与参考目标菌株在膜上成特异性杂交,对52个参考菌株和野外分离株的检测准确率为92%;膜杂交的敏感性与玻片芯片接近,最小检出量为100 fg DNA;在尼龙膜上增加K88探针,与3重PCR产物杂交,可以检测到大肠杆菌K88毒力基因。结论:建立的反向斑点杂交方法简便快速,检测成本低,可用于仪器设备不足的实验室,同时可以加入检测如大肠杆菌K88等致病基因,提高基于保守基因的芯片的诊断能力。     

Suspension arrays for high throughput, multiplexed single nucleotide polymorphism genotyping

BACKGROUND: Genetic diversity can help explain disease susceptibility and differential drug response. The most common type of variant is the single nucleotide polymorphism (SNP). We present a low-cost, high throughput assay for SNP genotyping. METHODS: The assay uses oligonucleotide probes covalently attached to fluorescently encoded microspheres. These probes are hybridized directly to fluorescently labeled polymerase chain reaction (PCR) products and the results are analyzed in a standard flow cytometer. RESULTS: The genotypes determined with our assay are in good agreement with those determined by TaqMan. The range of G/C content for oligonucleotide probes was 23.5-65% in the 17 bases surrounding the SNP. Further optimization of probe length and target concentration is shown to dramatically enhance the assay performance for certain SNPs. Using microspheres which have unique fluorescent signatures, we performed a 32-plex assay where we simultaneously determined the genotypes of eight different polymorphic genes. CONCLUSIONS: We demonstrate, for the first time, the feasibility of multiplexed genotyping with suspension arrays using direct hybridization analyses. Our approach enables probes to be removed from or added to an array, enhancing flexibility over conventional chips. The ability to multiplex both the PCR preparation and the hybridization should enhance the throughput, cost, and speed of the assay.     

Label-free and high-sensitive detection of Salmonella using a surface plasmon resonance DNA-based biosensor

A method based on surface plasmon resonance (SPR) DNA biosensor has been developed for label-free and high-sensitive detection of Salmonella. A biotinylated single-stranded oligonucleotide probe was designed to target a specific sequence in the invA gene of Salmonella and then immobilized onto a streptavidin coated dextran sensor surface. The invA gene was isolated from bacterial cultures and amplified using a modified semi-nested asymmetric polymerase chain reaction (PCR) technique. In order to investigate the hybridization detection, experiments with different concentration of synthetic target DNA sequences have been performed. The calibration curve of synthetic target DNA had good linearity from 5 nM to 1000 nM with a detection limit of 0.5 nM. The proposed method was applied successfully to the detection of single-stranded invA amplicons from three serovars of Salmonella, i.e., Typhimurium, Enterica and Derby, and the responses to PCR products were related to different S. typhimurium concentrations in the range from 10(2) to 10(10) CFU mL(-1). While with this system to detect E. coli and S. aureus, no significant signal was observed, demonstrating good selectivity of the method. In addition, the hybridization can be completed within 15 min, and the excellent sensor surface regeneration allows at least 300 assay cycles without obvious loss of performance.     

Rapid genotyping of the osteoporosis-associated polymorphic transcription factor Sp1 binding site in the COL1A1 gene by pyrosequencing

We describe a novel polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) sequencingbased assay for rapid genotyping of the polymorphic Sp1 binding site in the COL1A1 gene (1). A single nucleotide G-->T substitution polymorphism at this GC-rich site has recently been reported to be a predictive genetic marker for low bone mineral density (BMD). To simplify screening for this marker, we optimized PCR conditions and subjected the amplicons to pyrosequencing, which is a convenient high-throughput sequence analysis technique, readily amenable to automation. The analysis of 200 deidentified convenience DNA samples extracted from blood revealed genotype frequences in Hardy-Weinberg equilibrium (SS 68.0%, Ss 28.5%, and ss 3.5%) in agreement with other studies of European populations. This study demonstrates for the first time that pyrosequencing can be used for rapid identification of the osteoporosis-associated single nucleotide polymorphism (SNP) in the COL1A1 gene.     

Electrochemical measurement of DNA hybridization using nanosilver as label and horseradish peroxidase as enhancer

A simple and sensitive electrochemical DNA biosensor based on in situ DNA amplification with nanosilver as label and horseradish peroxide (HRP) as enhancer has been designed. The thiolated oligomer single-stranded DNA (ssDNA) was initially directly immobilized on a gold electrode, and quartz crystal microbalance (QCM) gave the specific amount of ssDNA adsorption of 6.3 ± 0.1 ng/cm². With a competitive format, hybridization reaction was carried out via immersing the DNA biosensor into a stirred hybridization solution containing different concentrations of the complementary ssDNA and constant concentration of nanosilver-labeled ssDNA, and then further binding with HRP. The adsorbed HRP amount on the probe surface decreased with the increment of the target ssDNA in the sample. The hybridization events were monitored by using differential pulse voltammetry (DPV) with the adsorbed HRP toward the reduction of H₂O₂. The reduction current from the enzyme-generated product was related to the number of target ssDNA molecules in the sample. A detection of 15 pmol/L for target ssDNA was obtained with the electrochemical DNA biosensor. Additionally, the developed approach can effectively discriminate complementary from non-complementary DNA sequence, suggesting that the similar enzyme-labeled DNA assay method hold great promises for sensitive electrochemical biosensor applications.     

Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material

A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pm sensitivity each consuming only 1 μl of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.     

Multiplexed SNP genotyping using the Qbead system: a quantum dot-encoded microsphere-based assay

We have developed a new method using the Qbead system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral 'barcodes' are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein-protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications.     

In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens

A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite-epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.     

DNA probes specific for Aeromonas hydrophila (HG1)

Aeromonas hydrophila (HG1)-specific RAPD-PCR fragments were investigated for their potential as DNA probes. From 20 RAPD-PCR fragment bands, it was found that two were specific to all isolates of Aeromonas hydrophila (HG1) tested. Cloning and nucleotide sequence determination of one of these bands showed that co-migration of similar sized amplicons had occurred and that this band (designated '7e') contained at least four fragments of different sequences. Three of these individual amplicons had a sequence specific to Aer. hydrophila (HG1) isolates. The sequence of one of these amplicons ('7e5') was used to design primers for a specific polymerase chain reaction (PCR). The specificity of the PCR was achieved using a modified hot-start procedure. The identity of the PCR amplicons was confirmed by high stringency hybridization with a digoxygenin-labelled 7e5 probe.