Multiple DNA Targets Research Articles

Молекулярные методы аутентификации молочного сырья в сыре: обзор

The modern dairy industry needs authentic raw milk in cheese production to provide high-quality product control and consumer protection. Unfortunately, unreliable cheese producers sometimes replace goat’s or sheep’s milk with cow’s milk. This process is associated with seasonality and the high cost of milk from small ruminants. The article reviews the methods molecular genetics used to detect milk adulteration in cheese making. It highlights the problems and prospects of applying molecular genetics to complex food matrices, such as cheese. Modern PCR methods prove quite efficient in identifying species-specific sequences by using mitochondrial DNA (mtDNA) as markers. Multiplex PCR, quantitative PCR (qPCR), and digital droplet PCR (ddPCR) have their advantages and shortcomings. The multiplex PCR method presupposes a simultaneous amplification of multiple DNA targets in a single tube while the qPCR method provides a quantitative profile. These methods make it possible to maintain high industry standards by ensuring the authenticity of cheeses and cheese products. In addition, molecular methods provide supply chain stability, increase consumer confidence, and advance dairy technology.

Programmable RNA detection with CRISPR-Cas12a

Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.

Rapid Multiplex PCR for Species Authentication: Current Prospects and Challenges

Multiplex PCR is a powerful technique that allows simultaneous amplification of multiple DNA targets in a single reaction. It has been widely applied for species authentication in various food matrices, such as meat, fish, dairy, and honey products. Species authentication is important for ensuring food safety, quality, traceability, and compliance with religious, ethical, and environmental standards. However, multiplex PCR also faces some challenges and limitations, such as primer design, optimization, validation, interference, and interpretation of results. This review summarizes the current state of the art of multiplex PCR for species authentication, highlighting its advantages, applications, challenges, and future directions. It also provides some practical guidelines and recommendations for designing and performing multiplex PCR assays for reliable and accurate species identification.

Next generation sequencing reveals targetable mutations in multiple sarcoma histologies.

11548 Background: Sarcomas are a rare and heterogenous group of cancers that arise from bone or soft tissue, and two thirds have poorly defined mutational profiles. Given their rarity, few comprehensive studies have fully characterized mutations and gene expression across sarcoma histologies correlated with clinical outcomes. Further studies are needed to determine the presence of targetable mutations that may improve patient outcomes. In this study, we explored the genomic landscape and clinical actionability of sarcoma mutations from patients enrolled in our CAUSAL (Cohort to Augment the Understanding of Sarcoma survivorship Across the Lifespan) study. Methods: Between 04/01/2022 and 01/01/2023, 481 participants, treated from 2012 – present with multiple sarcoma histologies were enrolled on CAUSAL. Next Generation Sequencing (NGS) was performed on primary or metastatic tumors from 76 patients to determine DNA mutations within a 648 gene panel. Whole transcriptome RNA sequencing (seq) provided expression profiles and RNA fusion products. Further analysis was performed using principal components analysis of RNA seq data to explore correlates amongst sarcoma subtypes. Tumor mutations were queried in ClinVar for relevance to known variants and were assigned to tiers I-IV based on the ESMO scale for clinical actionability of molecular targets (ESCAT). Tier I mutations have drug-mutation matched evidence of actionability while tier IV have only pre-clinical evidence. Results: NGS has been completed on 76 tumor samples. Sequenced tumors represented 19 histologies, with the most common ones as follows: undifferentiated pleomorphic sarcoma (15.8%), liposarcoma (9.2%), gastrointestinal stromal tumor (9.2%), osteosarcoma (7.9%), and leiomyosarcoma (7.9%). Of 76 patients, 66 (87%) had at least one mutation detected with an mean frequency of 2.74. TP53 (20/66), RB1 (14/66), and ATRX (9/66) were most commonly mutated genes. Mean (std) tumor mutation burden (TMB) was 3.4 m/MB (3.5m/MB) and one tumor had TMB of 25.3 m/MB. Of the 76 samples, 68 had RNA expression and fusion data available, of which 42 (62%) had anomalous expression changes and 11 had RNA fusions. The most overexpressed genes were NY-ESO-1 (13), LAGE-1 (9), and RET (7); the most under-expressed genes were SMARCB1 (9) and MGMT (6). 30.2% (23 of 76) of patients had potentially actionable DNA mutations, 9 had ESCAT tier I DNA mutations, 3 had tier II, 10 had tier III, and 1 had tier IV. 29.4% (20/68) of patients had potential targets based on RNA expression. 51.3% (39/76) patients had either a potential DNA or RNA target, and 6.6% (5/76) had multiple RNA or DNA targets. Conclusions: NGS revealed potentially actionable targets in over half of sarcoma patients based on ESCAT criteria. With ongoing accrual and sequencing of additional tumor specimens future analysis of CAUSAL will focus on assessing the correlation between targetable mutations and clinical outcomes.

Surface Functionalization and Reversible Disassembly of DNA-Assembly Nanoparticles for Sensitive and Multiplexed Detection of DNA Targets Without Enzymatic and Catalytic Amplification.

Recently, DNA-assembly nanoparticles based on DNA-metal ion interactions are emerging as new building blocks for drug delivery and metal nanostructure synthesis. However, the surface modification of DNA-assembly nanoparticles using functional biomolecules that can identify specific targets has rarely been explored. In this study, we developed a new immobilization chemical strategy to efficiently functionalize the barcode DNA-assembly nanoparticles (bcDNA NPs) with thiolated probe DNA (pDNA) for synthesizing pDNA-functionalized bcDNA NPs (pDNA-bcDNA NPs). We used them as nanoprobes to successfully demonstrate the sensitive and selective detection of multiple DNA targets. Importantly, Au ions played an essential role as anchoring sites via their conjugation with both thiolated pDNA and bcDNA NPs. In addition, we could reversibly and rapidly disassemble the pDNA-bcDNA NPs into the initial bcDNA strands with a recovery rate of 91%; this process significantly amplified the signal by releasing a million bcDNA strands, which enabled DNA quantification from a single pDNA-bcDNA NP. The Au3+ concentration, pH, and surface passivation conditions were carefully investigated to maximize the pDNA loading to 8500 strands/bcDNA NP. The limit of detection was determined to be 221 fM, which is the most sensitive among the absorbance-based methods without polymerase chain reaction, hybridization chain reactions, catalytic hairpin assembly, and other reactions involving enzymes and catalysts. The reversible disassembly of DNA strands and Au ion-mediated conjugation chemistry could be extended for the detection of other types of targets, such as proteins, metal ions, and small molecules, using other organic functionalities that are or can be thiolated, including polypeptides, aptamers, and antibodies.

Structural basis of DNA binding by the NAC transcription factor ORE1, a master regulator of plant senescence

Plants use sophisticated mechanisms of gene expression to control senescence in response to environmental stress or aging. ORE1 (Arabidopsis thaliana NAC092) is a master regulator of senescence that belongs to the plant-specific NAC transcription factor protein family. ORE1 has been reported to bind to multiple DNA targets to orchestrate leaf senescence, yet the mechanistic basis for recognition of the cognate gene sequence remains unclear. Here, we report the crystal structure of the ORE1–NAC domain alone and its DNA-binding form. The structure of DNA-bound ORE1–NAC revealed the molecular basis for nucleobase recognition and phosphate backbone interactions. We show that local versatility in the DNA-binding site, in combination with domain flexibility of the ORE–NAC homodimer, is crucial for the maintenance of binding to intrinsically flexible DNA. Our results provide a platform for understanding other plant-specific NAC protein–DNA interactions as well as insight into the structural basis of NAC regulators in plants of agronomic and scientific importance.

A high-throughput analytical method for multiple DNA targets based on microdroplet PCR coupled with DGGE

We developed a novel approach to analyze multiple DNA targets based on microdroplet PCR coupled with denaturing gradient gel electrophoresis (MPDG) to achieve high-throughput detection of biological samples. The target DNAs were preamplified using specific primers. Subsequently, the preamplified products were separated into individual microreactors for parallel amplification with high efficiency, avoiding the interference of different primers and templates, and preventing inconsistent amplification efficiency and non-specific amplification. The final products were analyzed using denaturing gradient gel electrophoresis (DGGE). Using genetically modified (GM) maize as samples, the MPDG method could be used for the simultaneous detection of three DNA targets with an absolute limit of detection of 0.5% (w/w), with no cross reaction with other non-GM samples. The simulated sample assay of MPDG suggested that the method is suitable for practical application. The MPDG approach, with high sensitivity and specificity, could play a crucial role in the field of nucleic acid detection.

Highly multiplexed rapid DNA detection with single-nucleotide specificity via convective PCR in a portable device.

Assays for the molecular detection of nucleic acids are typically constrained by the level of multiplexing (this is the case for the quantitative polymerase chain reaction (qPCR) and for isothermal amplification), turnaround times (as with microarrays and next-generation sequencing), quantification accuracy (isothermal amplification, microarrays and nanopore sequencing) or specificity for single-nucleotide differences (microarrays and nanopore sequencing). Here we show that a portable and battery-powered PCR assay performed in a toroidal convection chamber housing a microarray of fluorescently quenched oligonucleotide probes allows for the rapid and sensitive quantification of multiple DNA targets with single-nucleotide discrimination. The assay offers a limit of detection of 10 DNA copies within 30 min of turnaround time and a dynamic range spanning 4 orders of magnitude of DNA concentration, and we show its performance by detecting 20 genomic loci and 30 single-nucleotide polymorphisms in human genomic DNA samples, and 15 bacterial species in clinical isolates. Portable devices for the fast and highly multiplexed detection of nucleic acids may offer advantages in point-of-care diagnostics.

Dual-color graphene quantum dots and carbon nanoparticles biosensing platform combined with Exonuclease III-assisted signal amplification for simultaneous detection of multiple DNA targets

Sensitive and simultaneous detection of multiple biomarkers such as target DNA or proteins using biocompatible materials with good analysis performance remains an important challenge. Herein, we successfully developed a signal “off-on” highly sensitive multiplex detection platform based on the combination of dual-color graphene quantum dots (blue GQDs and green GQDs) modified DNA probes with carbon nanoparticles (CNPs), which is a cheap, effective nonfluorescent quencher to simultaneously quench the fluorescence of both GQDs-DNA probes. The Exo III-assisted sequence-independent target recycling and signal amplification strategy was integrated into this sensing platform, which endows it with high sensitivity towards the multiplex detection of targets DNA. The detection limits of 6.6 pM for HIV and 9.5 pM for HBV were achieved respectively, which is about 60-fold lower than that of traditional unamplified homogeneous fluorescent assay methods. Our proposed multiplex detecting platform is advantageous in both respective and simultaneous detection of multiple targets and can also discriminate perfectly matched targets from mismatched targets in both PBS buffer and 1% human serum samples, demonstrating its potential to be a reliable strategy for highly sensitive simultaneous detection of multiple target genes in practical diagnosis applications.

Busting the myths: DNA typeability after 48 hours of boil

The aim of our study was to monitor the quality and quantity of DNA in bone samples that were boiled for 48 h. Bos taurus bone disks were sampled every hour for 48 h. The subsequent DNA analysis used multiple mitochondrial DNA (mtDNA) targets (100–700 bp) to evaluate the quality and quantity of the DNA extracted. The DNA extracted from bone disks remained typeable after boiling for 48 h. We have proven that DNA typing results can be obtained even after long-term boiling.

Multiplexed DNA detection with DNA tweezers in a one-pot reaction

One of the great challenges for the development of biosensors is to construct a fast, practical and multiplexed detection platform. One promising approach is utilizing the controllable conformation of DNA nanostructures for such biosensing applications. Here, we present tweezer-like DNA (TW-DNA)-assisted multiplexed DNA detection in a one-pot reaction based on fluorescence quenching. The open conformation of the TW-DNA structure can be changed to a closed state by a specific single-stranded DNA target. The fluorophore/quencher pairs decorated on the TW-DNA and the conformation changes were observed by fluorescence quenching. In its initial state, the TW-DNA is maintained in an open state that demonstrates high fluorescence intensity. In the presence of a specific target DNA, the TW-DNA changes to a closed conformation and the emission intensity of the donor is quenched which is directly proportional to the target concentration. Three DNA targets were simultaneously detected using three individually self-assembled TW-DNA that were modified appropriately with fluorophore/quencher pairs for the specific detection of the individual DNA target. The detection range of this assay is at submicromolar concentration of multiple DNA targets with a fast detection time of one minute. Moreover, this biosensor platform provides the significant advantage of being a one-pot reaction for multiplexed DNA detection without any cross-talk, thereby making it a highly applicable assay.

A novel emulsion PCR method coupled with fluorescence spectrophotometry for simultaneous qualitative, quantitative and high-throughput detection of multiple DNA targets

We constructed and validated a novel emulsion PCR method combined with fluorescence spectrophotometry (EPFS) for simultaneous qualitative, quantitative and high-throughput detection of multiple DNA targets. In a single reaction set, each pair of primers was labeled with a specific fluorophore. Through emulsion PCR, a target DNA was amplified in droplets that functioned as micro-reactors. After product purification, different fluorescent-labeled DNA products were qualitatively analyzed by the fluorescent intensity determination. The sensitivity and specificity of the system was examined using four kinds of genetically modified (GM) maize. The qualitative results revealed high specificity and sensitivity of 0.5% (w/w). In addition, the quantitative results revealed that the absolute limit of detection was 103 copies, showing good repeatability. Moreover, the reproducibility assays were further performed using four foodborne pathogenic bacteria to further evaluate the applicability of the system. Consequently, the same qualitative, quantitative and high-throughput results were confirmed with the four GM maize. To sum up, the new EPFS system is the first analytical technology of this kind that enables simultaneous qualitative, quantitative and high-throughput analysis of multiple genes.

Abstract 941: Method for sensitive detection of tumor fingerprints in plasma

Abstract Presence of excess unaltered, wild-type DNA providing no information of biologic or clinical value may often mask rare alterations containing diagnostic or therapeutic clues for cancer. There is a growing demand for removing unaltered DNA over large pools-of-sequences, especially for applications in liquid biopsies with circulating DNA (cfDNA). We recently developed nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO), a single-step approach that removes WT-DNA prior to DNA amplification, following which current genomic analysis processes remain unchanged. NaME-PrO employs a double-strand DNA-specific nuclease (DSN) and overlapping oligonucleotide-probes interrogating multiple DNA targets. Following genomic-DNA denaturation, the temperature is lowered and the probes form transient double-stranded regions with their targets, and DSN is added to provide nuclease digestion to the selected sites. Mutations create mismatches that inhibit DSN digestion; thus, subsequent amplification yields DNA with alterations enhanced at multiple targets. In this manner, WT DNA at hundreds or thousands of DNA regions can be digested simultaneously. We also show that addition of organic solvents like DMSO in the NaME-PrO reaction enables DNA denaturation at lower temperatures that do not inactivate the enzyme (DSN). This enables DSN addition from the beginning of the reaction, enabling homogeneous, closed-tube application of the approach and avoids contamination-prone sample manipulations due to open-tube addition of DSN enzyme after denaturation. Using NaME-PrO ± DMSO we demonstrate several-hundred-fold multiplexed mutation enrichment in diverse human samples on multiple clinically relevant targets in 50-plex reactions. Application with targeted resequencing of tumor-circulating DNA demonstrated detection of mutations at the 0.01-0.1% levels with few sequence reads. In ongoing studies aiming to detect minimal residual disease using cfDNA in late-stage breast cancer, we used tumor exome sequencing to identify multiple clonal mutations for individual patients. We then performed multiplexed NaME-PrO to trace the "tumor fingerprint" in plasma. In ~50-plex reactions we showed that the majority (>60%) of the tumor-specific mutations were also detected in plasma. To evaluate the ultimate sensitivity of the method to identify such "tumor fingerprints," we applied NaME-PrO in serial dilutions of cfDNA from breast cancer patients into cfDNA from cancer-free, normal patients. We demonstrated that dilutions down to 1,000-10,000-fold, corresponding to mutations at <0.01% allelic frequency, lead to identification of at least one tumor-specific mutation in plasma. NaME-PrO can be combined with platforms that utilize molecular barcodes to provide "maximum efficiency sequencing," enabling sensitive detection of low-level mutations without requiring "deep sequencing" and excessive sequence reads. Citation Format: Fangyan Yu, Mariana Fitarelli-Kiehl, Ka Wai Leong, Viktor Adalsteinsson, Heather Parsons, Mike G. Makrigiorgos. Method for sensitive detection of tumor fingerprints in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 941.

Nonaplex PCR using Cliffhanger primers to identify diarrhoeagenic Escherichia coli from crude lysates of human faecal samples.

Sensitive, probe-based detection of multiple DNA targets is limited by the competitive reannealing of the antiparallel duplex DNA helix with the complementary DNA strand. To address this, we developed Cliffhanger primers, which create single-stranded DNA overhangs on PCR amplicons while simultaneously increasing the multiplex PCR efficacy and allowing PCR amplification using crude lysates of human faecal samples. A multiplex PCR that targeted eight genes from diarrhoeagenic Escherichia coli plus an internal control was performed and compared to a routine method that consisted of culture followed by multiplex PCR with fragment length separation. A total of 2515 clinical faecal samples from patients with diarrhoea were tested using both methods, and there was a significant increase in clinical sensitivity and negative predictive value with the Cliffhanger method for seven out of eight genes. All Cliffhanger-only positive samples were confirmed by Sanger sequencing of the PCR amplicon. Notably, the Cliffhanger method reduced the total sample turn-around time in the laboratory from 20 hours to 6 hours. Hence, use of Cliffhanger primers increased assay robustness, decreased turn-around time and increased PCR efficacy. This increased the overall clinical sensitivity without the loss of specificity for a heavily multiplexed PCR assay.

Silver-dendrimer nanocomposites as oligonucleotide labels for electrochemical stripping detection of DNA hybridization

Silver-dendrimer nanocomposites were synthesized and used as oligonucleotide labels for electrochemical stripping detection of DNA hybridization. The synthesized silver-dendrimer nanocomposites were characterized by UV–vis spectrophotometry, X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). Ratios of silver/dendrimer were optimized in order to obtain stable nanocomposites with maximal silver loading in the interior of a polymeric shell. The silver-dendrimer nanocomposites were attached to sequence-known DNA probes specific to colitoxin, and used to detect probe hybridization by dissolution of the silver nanoparticles in the interior of dendrimer in a diluted nitric acid, followed by measurement of Ag+ ions by anodic stripping voltammetry (ASV). Use of differential pulse voltammetry for the stripping step, along with optimization of the ASV conditions, enabled a detection limit of 0.78 pM. The present strategy, in combination with dendrimer-encapsulated copper labeled oligonucleotides probe reported previously, could potentially be used to detect single or multiple DNA targets in one sample.

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies.

The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing. We developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA. Serial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 10-20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets. cfDNA mutations were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing. Closed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable prescreening before targeted resequencing.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets.

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 102-105 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection.

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.

Loading of silicon nanoparticle labels with redox mediators for detection of multiple DNA targets within a single voltammetric sweep

We have constructed electrochemical labels by loading four different redox mediators into silica nanoparticles (SiNPs) through electrostatic attraction. The mediators were methylene blue, ferrocinium tetrafluoroborate, tris (2,2-bipyridyl) dichlororuthenium (II) hexahydrate and acridine orange. The SiNPs were coated with avidin, which enabled attachment to biotin-tagged DNA sequences, forming signal probes. The immobilisation of four different capture probes onto screen printed electrodes enabled a simple qualitative and semi-quantitative detection of four different target probes within a single voltammetric sweep using differential pulse voltammetry. This was illustrated by detecting sequences found in four forms of the influenza virus, the influenza A sub-types H1N1, H3N2, H5N1 and influenza B. The limits of detection were 1.6pM, 1.6pM, 4.7pM and 2.9pM, respectively.

Multiplex Strand Invasion Based Amplification (mSIBA) assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

Nucleic acid amplification tests have become a common method for diagnosis of STIs due to their improved sensitivity over immunoassays and traditional culture-based methods. Isothermal nucleic acid amplification methods offer significant advantages over polymerase chain reaction (PCR) because they do not require sophisticated instruments needed for thermal cycling of PCR. We recently reported a novel isothermal nucleic acid amplification method, Strand Invasion-Based Amplification (SIBA), which exhibited high analytical sensitivity and specificity for amplification of DNA. However, because the reactions were detected using an intercalating dye, this method was only suitable for amplifying a single genomic target. Here, we report the development of multiplexed SIBA (mSIBA) that allows simultaneous detection of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and an internal control in the same reaction tube. SIBA is compatible with probes, allowing the detection of multiple DNA targets in the same reaction tube. The IC was developed to assess the quality of the isolated DNA and the integrity of the enzyme system, as well as to test oligonucleotides. The mSIBA assay retained high analytical sensitivity and specificity for the detection of CT and NG. The development of mSIBA enables rapid screening for CT and NG within point-of-care or central laboratory settings.