Nano-magnetic primer based electrochemiluminescence-polymerase chain reaction (NMPE-PCR) assay

Here we have developed a novel nano-magnetic primer based electrochemiluminescence-polymerase chain reaction (NMPE-PCR) strategy for detection of genome. The key idea of this method is integrating the two in situ processes: PCR on the surface of magnetic nanoparticles (MNPs) and magnetic beads based ECL readout platform, to avoid some laborious manual operations and achieve rapid yet sensitive detection. At first, the approach employs a pair of functional primers for amplification: one is tris-(2,2'-bipyridyl) ruthenium (TBR) labeled primer; the other one is nano-magnetic primer which is prepared by attaching the primer to the surfaces of MNPs. With the presence of DNA analyte and PCR mixture, the TBR labeled products are directly loaded and enriched on the surface of MNPs during PCR cycling. Then the MNPs-TBR complexes can be analyzed by a magnetic ECL platform without any post-modification or post-incubation. Finally, we used Listeria monocytogenes as the target to examine these desirable properties of this assay, reaching a detection limit of 500 fg/μL for genome in 1 h. The proposed study has provided the evidence as a proof-of-concept, thus having potential for development of automatic mode for detection of specific gene.     

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.     

Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay.

A new detection system, the magnetic immuno-polymerase chain reaction (PCR) assay (MIPA) has been developed to detect Listeria monocytogenes in food. This method separates Listeria cells from PCR-inhibitory factors present in enrichment broths containing food samples by using magnetic beads coated with specific monoclonal antibodies (MAbs). The separated bacteria were lysed, and the supernatant containing the bacterial DNA was subjected to the PCR. Detection of L. monocytogenes in three naturally contaminated cheese samples with two different MAbs and PCR primers specific for the gene encoding the delayed-hypersensitivity factor showed that with MAb 55 all three samples were positive whereas with MAb A two samples were positive. A further improvement of the method was obtained by using a PCR step based on the listeriolysin O gene. A MIPA employing MAb 55 and the listeriolysin O gene primer set detected L. monocytogenes after 24 h of culture in Listeria Enrichment Broth samples from Port Salut artificially contaminated with 40 CFU/25 g. We could detect 1 CFU of L. monocytogenes per g of cheese after a second enrichment for 24 h in Fraser broth. The analysis time including both enrichments is approximately 55 h.     

Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay

We report a duplex real-time PCR-based assay for the simultaneous quantitative detection of Listeria spp. and the food-borne pathogen Listeria monocytogenes. The targets of this single tube reaction were the 23S rDNA and hly genes of Listeria spp. and L. monocytogenes, respectively. Our assay was efficient, 100% selective (i.e., it allowed accurate simultaneous identification of 52 L. monocytogenes and 120 Listeria spp. strains through the FAM-labelled hly and the VIC-labelled 23S rDNA probes, respectively); and had a detection limit of one target molecule in 100% (23S rDNA) and 56% (hly) of the reactions. Simultaneous quantification was possible along a 5-log dynamic range, with an upper limit of 30 target molecules and R2 values > 0.995 in both cases. Our results indicate that this assay based on the amplification of the 23S rDNA gene can accurately quantify any mixture of Listeria species and simultaneously unambiguously quantify L. monocytogenes.     

A novel real-time PCR for Listeria monocytogenes that monitors analytical performance via an internal amplification control

We describe a novel quantitative real-time (Q)-PCR assay for Listeria monocytogenes based on the coamplification of a target hly gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed BnACCg8 gene flanked by the hly-specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the hly and IAC signals. The hly-IAC assay had a specificity and sensitivity of 100%, as assessed using 49 L. monocytogenes isolates of different serotypes and 96 strains of nontarget bacteria, including 51 Listeria isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity (R2) and efficiency (E) were 0.997 and 0.80, respectively. We tested the performance of the hly-IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for L. monocytogenes, the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure.     

Cocaine detection via rolling circle amplification of short DNA strand separated by magnetic beads

A novel and sensitive fluorescence biosensor based on aptamer and rolling circle amplification for the determination of cocaine was developed in the present work. Here cocaine aptamers immobilized onto Au nanoparticles modified magnetic beads hybridized with short DNA strand. In the presence of cocaine, the short DNA strand was displaced from aptamer owing to cocaine specially binding with aptamer. Next, the short DNA strand was separated by magnetic beads and used to originate rolling circle amplification as primer. The end products of rolling circle amplification were detected by fluorescence signal generation upon molecular beacons hybridizing with the end products of rolling circle amplification. With rolling circle amplification and the separation by magnetic beads reducing the background signal, the new strategy was suitable for the detection of as low as 0.48 nM cocaine. Compared with reported cocaine sensors, our method exhibited excellent sensitivity. Our new strategy may provide a platform for numerous proteins and low molecular weight analytes to highly sensitively detect by DNA amplification.     

Quantum dots-based multifunctional dendritic superstructure for amplified electrochemiluminescence detection of ATP

A novel multifunctional dendrimeric CdSe-CdS-Quantum dots (QDs) hybrid superstructure with highly intense electrochemiluminescence (ECL), fluorescence and excellent magnetic property is prepared for the first time, and successfully applied to amplified ECL assays of ATP using DNA cycle amplification technique. The magnetic nanoparticles (MNPs) were firstly assembled with unique dendrimer nanoclusters (NCs), then large numbers of QDs were labeled onto the dendrimer NCs, the superstructure exhibits highly enhanced ECL and fluorescence than the pure QDs. Remarkable ECL quenching of the nanocomposites by gold nanoparticles (GNPs) was observed, based on which a novel strategy for highly sensitive ATP detection was developed by cycle amplification technique. Furthermore, the nanocomposites with excellent magnetic properties can be easily labeled, separated and immobilized onto a magnetic electrode. In particular, all the procedures such as linking GNPs, sensing target and DNA cycle amplification were directly accomplished on the nanocomposites, which is more rapid, convenient, complete and has better reproducibility than the conventional methods on electrode. To the best of our knowledge, this is the first report on the multifunctional QDs superstructure with highly intense ECL, fluorescence, excellent magnetism and its ECL biosensing, which opens a new pathway for developing QD-based nanocomposites for broad applications in ECL bioassays and optical imaging.     

Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction.

A sensitive and specific method for detection of Listeria monocytogenes in milk and ground-beef samples is described. It consists of culturing samples in listeria enrichment broth (LEB) and subculturing them from LEB to listeria plating media, followed by DNA extraction and species-specific detection of the organism by using the polymerase chain reaction (PCR). In developing the L. monocytogenes PCR assay, five oligonucleotide primers complementary to the nucleotide sequence of the listeriolysin O gene were synthesized and used in amplification experiments. PCR products of the predicted size, based on nucleotide sequence information, were generated with DNA from all of 72 L. monocytogenes strains with five different primer pairs. DNA from Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeia murrayi strains and a panel of 47 bacterial strains representing 17 genera did not generate PCR products with the primer pairs employed. As little as 1 pg of L. monocytogenes DNA could be detected with the assay. To determine the most sensitive culture protocol to use in conjunction with the PCR assay, milk (10 ml) and ground-beef (25 g) samples were inoculated with L. monocytogenes at concentrations ranging from 0 to 10(5) CFU ml-1 or g-1, as appropriate for the sample. PCR assays on DNA extracted from growth on listeria plating media, inoculated with 24-h LEB samples cultures, were most sensitive, allowing detection of as little as 0.1 CFU of L. monocytogenes ml-1 or g-1 of milk and ground beef, respectively.     

Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction.

A sensitive and specific method for detection of Listeria monocytogenes in milk and ground-beef samples is described. It consists of culturing samples in listeria enrichment broth (LEB) and subculturing them from LEB to listeria plating media, followed by DNA extraction and species-specific detection of the organism by using the polymerase chain reaction (PCR). In developing the L. monocytogenes PCR assay, five oligonucleotide primers complementary to the nucleotide sequence of the listeriolysin O gene were synthesized and used in amplification experiments. PCR products of the predicted size, based on nucleotide sequence information, were generated with DNA from all of 72 L. monocytogenes strains with five different primer pairs. DNA from Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeia murrayi strains and a panel of 47 bacterial strains representing 17 genera did not generate PCR products with the primer pairs employed. As little as 1 pg of L. monocytogenes DNA could be detected with the assay. To determine the most sensitive culture protocol to use in conjunction with the PCR assay, milk (10 ml) and ground-beef (25 g) samples were inoculated with L. monocytogenes at concentrations ranging from 0 to 10(5) CFU ml-1 or g-1, as appropriate for the sample. PCR assays on DNA extracted from growth on listeria plating media, inoculated with 24-h LEB samples cultures, were most sensitive, allowing detection of as little as 0.1 CFU of L. monocytogenes ml-1 or g-1 of milk and ground beef, respectively.     

Detecting nucleic acid fragments in serum using a magnetically modulated sandwich assay

We present a novel assay for rapid and highly sensitive detection of specific nucleic acid fragments in human serum. In a magnetic modulation biosensing (MMB) system, magnetic beads and fluorescently labeled probes are attached to the target analyte and form a “sandwich” complex. An alternating external magnetic field gradient condenses the magnetic beads (and hence the target molecules with the fluorescently labeled probes) to the detection volume and sets them in a periodic motion, in and out of a laser beam. A synchronous detection enables the removal of background signal from the oscillating target signal without complicated sample preparation. The high sensitivity of the MMB system, combined with the specificity of a sandwich hybridization assay, enables detection of DNA fragments without enzymatic signal amplification. Here, we demonstrate the sensitivity of the assay by directly detecting the EML4‐ALK oncogenic translocation sequence spiked in human serum. The calculated limit of detection is 1.4 pM, which is approximately 150 times better than a conventional plate reader. In general, the MMB‐assisted SHA can be implemented in many other applications for which enzymatic amplification, such as PCR, is not applicable and where rapid detection of specific nucleic acid targets is required.     

Hyperbranched rolling circle amplification as a novel method for rapid and sensitive detection of Amphidinium carterae

High quality of coastal water is critical to marine ecosystems, marine fisheries, public health, and aquatic environment. Specially, bio-toxin derived from toxic microalgae is currently threatening many coastal countries. Therefore, development of rapid and sensitive methods for the detection of toxin-producing microalgae is necessary for warning of water quality. In this paper, we established a novel method for rapid and sensitive detection of Amphidinium carterae by hyperbranched rolling circle amplification (HRCA). The partial large subunit rDNA (LSU D1–D2) of A. carterae was sequenced to design species-specific padlock probe (PLP). The PLP-coupled with two amplification primers were employed for HRCA. The optimized HRCA conditions were as follows: padlock concentration, 20 pM; ligation temperature, 65 °C; ligation time, 15 min; amplification temperature, 61 °C; and amplification time, 15 min. The developed HRCA was confirmed to be specific for A. carterae by tests with other algae. The sensitivity of HRCA was 100-fold higher than regular PCR, exhibiting a detection limit of 1 fg/μL representing 283 copies for the recombinant plasmid containing the target LSU D1–D2, and 1 cell for target species. Finally, a simplified protocol was applied to the simulated field and environmental materials, and exhibited a good performance. The whole detection could be completed within 1.5 h, displaying a repeated detection limit of 1 cell. The positive HRCA results could be visualized through coloration reaction by adding the fluorescent dye SYBR Green I to the amplification products. The HRCA provides a useful tool to quickly screen large sample sets for A. carterae, as well as other toxic species.     

Detection of Prorocentrum minimum (Pavillard) Schiller with an Electrochemiluminescence–Molecular Probe Assay

The occurrence of harmful algal blooms (HABs) caused by Prorocentrum minimum (Pavillard) Schiller is a crucial subject in the study of HABs. An electrochemiluminescence-molecular probe assay (ECL-MP) was developed to qualitatively and quantitatively detect P. minimum. It was based on the sandwich hybridization integrated with a nuclease protection assay (NPA-SH) and improved by electrochemiluminescence (ECL). An ECL analyzer was established in this study, and it was shown that this analyzer was stable and highly sensitive, with a detection range of 0.4?pmol to 4?nmol Ru(bpy)(3)Cl(2)·6H(2)O under optimal reaction conditions of 1.0?V, 1.0?mA, 1.5?mol·L(-1) TPrA, and pH?7.4. The optimal amount of magnetic beads for separation of labeled NPA probes in a 20-μL hybridization mixture was 4?μg. The ECL counts per second was linear with the number of P. minimum cells in a range of 6.25?×?10(2) to 4?×?10(4), and there was no significant difference between ECL-MP and microscopy, with a 95% confidence level (t test) when individual, mixed cultures and field samples were treated. This study provides a convenient method for fast and accurate detection of P. minimum in the marine environment.     

Novel multiplex PCR approaches for the simultaneous detection of human pathogens: Escherichia coli 0157:H7 and Listeria monocytogenes

Escherichia coli 0157:H7 and Listeria monocytogenes are the two most important food-borne human pathogens. To develop a single, rapid and sensitive PCR based test for simultaneous detection of both the organisms, fliCh7 and iap gene specific primers were used respectively for E. coli 0157:H7 and L. monocytogenes. Initially, with equal quantities of purified genomic DNAs of these organisms a multiplex PCR reaction was standardized to yield uniform amplification of both targets. Although, this assay detected E. coli 0157:H7 with high sensitivity, it failed to pick up L. monocytogenes after several hours of enrichment in broth medium initially spiked with equal numbers of live cells. This was found to be due to unequal growth of these organisms leading to disparity in the amount of template DNAs represented in the DNA preparation applied for conventional multiplex PCR amplification. To circumvent this, we have developed a modified method of enrichment and harvesting leading to highly sensitive and rapid single reaction PCR detection of both pathogens. We have also successfully developed two novel multiplex PCR formats for the generation of uniform PCR signals. Some of these methods might find broader application for the simultaneous detection of different combinations of multiple pathogens.     

Highly sensitive protein kinase activity assay based on electrochemiluminescence nanoprobes

Herein, we describe a novel electrochemiluminescence (ECL) biosensor for protein kinase activities and inhibition monitoring based on the magnetic beads (MB) technology and signal enhancement of gold nanoparticles (GNP). In this design, ECL nanoprobes were prepared by conjugating GNP with phosphorylated DNA capture probes and tris-(2,2'-bipyridyl) ruthenium (TBR)-cysteamine. Zirconium cations, a specific bridging agent, mediate the linkage between biotin modified phosphorylated peptides and ECL nanoprobes. The complexes were then captured and enriched on the electrode surface by streptavidin-coated MB for ECL reaction. To confirm the feasibility of this biosensor, we employed protein kinase A (PKA) as the model kinase to validate the assay and a satisfactory detection limit of 0.005 U/mL was achieved. The combination of ECL and GNP lays a solid foundation for highly sensitive assay, meanwhile, the coupling of MB surfaces used for separation and capture with unmodified ECL electrode detection results in a greatly simplified and reusable protocol. Thus, our biosensor offers great promise for a highly sensitive and simple assay for protein kinase activity. Furthermore, the inhibition of PKA activity was monitored on the basis of the ECL signals change in response to the concentration of PKA inhibitor.     

4-(dimethylamino)butyric acid@PtNPs as enhancer for solid-state electrochemiluminescence aptasensor based on target-induced strand displacement

A solid-state electrochemiluminescence (ECL) aptasensor based on target-induced aptamer displacement for highly sensitive detection of thrombin was developed successfully using 4-(dimethylamino)butyric acid (DMBA)@PtNPs labeling as enhancer. Such a special aptasensor included three main parts: ECL substrate, ECL intensity amplification and target-induced aptamer displacement. The ECL substrate was made by modifying the complex of Pt nanoparticles (PtNPs) and tris(2,2-bipyridyl) ruthenium (II) (Ru(bpy)(3)(2+)) (Ru-PtNPs) onto nafion@multi-walled carbon nanotubes (nafion@MWCNTs) modified electrode surface. A complementary thrombin aptamer labeled by DMBA@PtNPs (Aptamer II) acted as the ECL intensity amplification. The thrombin aptamer (TBA) was applied to hybridize with the labeled complementary thrombin aptamer, yielding a duplex complex of TBA-Aptamer II on the electrode surface. The introduction of thrombin triggered the displacement of Aptamer II from the self-assembled duplex into the solution and the association of inert protein thrombin on the electrode surface, decreasing the amount of DMBA@PtNPs and increasing the electron transfer resistance of the aptasensor and thus resulting large decrease in ECL signal. With the synergistic amplification of DMBA and PtNPs to Ru(bpy)(3)(2+) ECL, the aptasensor showed an enlarged ECL intensity change before and after the detection of thrombin. As a result, the change of ECL intensity has a direct relationship with the logarithm of thrombin concentration in the range of 0.001-30 nM. The detection limit of the proposed aptasensor is 0.4 pM. Thus, the approach is expected to open new opportunities for protein diagnostics in clinical as well as bioanalysis in general.     

Nucleic acid sequence based amplification for the rapid and sensitive detection of Salmonella enterica from foods

AIMS: The purpose of this study was to apply nucleic acid sequence-based amplification (NASBA) for the detection of Salmonella enterica serovar Enteritidis (S. Enteritidis) in representative foods. METHODS AND RESULTS: A previously reported primer and probe set based on mRNA sequences of the dnaK gene of Salmonella were used in this study. To test for possible food matrix inhibition and assay detection limits, 25-g samples of representative food commodities (fresh meats, poultry, fish, ready-to-eat salads and bakery products) were pre-enriched with and without S. Enteritidis inoculation. The NucliSens(R) Basic Kit, supplemented with enzymes from various other commercial sources, was used for RNA isolation, NASBA amplification and electrochemiluminescent (ECL) detection. The end point detection limit of the NASBA-ECL assay was equivalent to 101 CFU of S. Enteritidis per amplification reaction. When the assay was tested on noncontaminated foods, none of the food matrices produced false-positive results. Some of the food matrices inhibited the NASBA-ECL reaction unless the associated RNA was diluted 10-fold prior to amplification. CONCLUSIONS: For all food items tested, positive ECL signals were achieved after 18 h of pre-enrichment and subsequent NASBA at initial inoculum levels of 102 and 101 CFU per 25 g food sample. SIGNIFICANCE AND IMPACT OF THE STUDY: This rapid, semi-automated detection method has potential for use in the food, agricultural and public health sectors.