Hybridization-based Detection Research Articles

Development of a Simple Method to Detect the Carbapenemase-Producing Genes blaNDM, blaOXA-48-like, blaIMP, blaKPC, and blaVIM Using a LAMP Method with Lateral Flow DNA Chromatography.

Infections by carbapenemase-producing Enterobacterales constitute a global public health threat. The rapid and efficient diagnosis of Enterobacterales infection is critical for prompt treatment and infection control, especially in hospital settings. We developed a novel loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography to identify five major groups of carbapenemase-producing genes (blaNDM, blaOXA-48-like, blaIMP, blaKPC, and blaVIM). This method uses DNA-DNA hybridization-based detection in which LAMP products can be easily visualized as colored lines. No specific technical expertise, expensive equipment, or special facilities are required for this method, allowing its broad application. Here, 73 bacteria collections including strains with carbapenemase-producing genes were tested. Compared to sequencing results, LAMP DNA chromatography for five carbapenemase-producing genes had a sensitivity and specificity of 100% and >97%, respectively. This newly developed method can be a valuable rapid diagnostic test to guide appropriate treatments and infection control measures, especially in resource-limited settings.

Nucleic acid hybridization-based detection of pathogenic RNA using microscale thermophoresis

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2h, demonstrating its diagnostic potential for early pathogen detection.

Synthetic Receptor Based on a Peptide Antibiotic-Functionalized Chimera for Hybridization-Based Polynucleotide Detection.

Nanopores offer highly sensitive, low-cost, and single-molecule sensing capabilities, and the societal impact of this approach is best captured by the advent of nanopore-based DNA detection and sequencing technologies, which extract genomic information without amplification. To address a critical difficulty plaguing such undertakings involving especially protein-based nanopores isolated in lipid bilayers, namely, the formation of a stable, long-lasting single nanopore, we pioneer herein an approach for generating functional nanostructures enabling small single-stranded DNA (ssDNA) detection. We designed a dynamic hybrid construct by appending extramembrane peptide nucleic acid (PNA) segments to the C-terminus of modified ion channel-forming alamethicin monomers. We found that the resulting chimeric molecules successfully coassemble in a voltage-dependent manner in planar lipid membranes generating diameter-variable oligomers. The subsequent interaction at the flexible extramembrane segment of such formed dynamic nanopores with aqueously added complementary ssDNA fragments leads to overall conformational alterations affecting the peptide assembly state kinetics and mediated ionic current. Such recognition events were found specific to the primary structure of target ssDNA and uninhibited the presence of serum. Our platform demonstrates the feasibility of designing an entirely new class of versatile chimeric biosensors, for which, dependent upon the nature of the attached receptor moiety and underlying recognition chemistry, the applicability area may extend to other analytes.

PCR-dipstick DNA Chromatography for detection of Carbapenemase Producing Carbapenem-Resistant Enterobacteriaceae

Carbapenemase producing carbapenem-resistant Enterobacteriaceae (CP-CRE) are an epidemiologically important emerging public health threat. Early and reliable detection of CP-CRE is important for containment of these pathogens. The aim of this study was to evaluate and observe the sensitivity of polymerase chain reaction (PCR)-dipstick DNA chromatography (GeneFields CPE) for detection of carbapenemase genes (NDM, OXA-48, KPC, IMP) in comparison to conventional PCR. In this cross-sectional study, a total of 145 carbapenem resistant Enterobacteriaceae isolates were subjected to test for carbapenemase by phenotypic mCIM and genotypic- conventional PCR and PCR-dipstick DNA Chromatography method. In PCR dipstick DNA chromatography assay, after multiplex PCR a DNA-DNA hybridization-based detection system is applied where amplified products can be easily interpreted by observing a blue line in DNA strip visually within 15 minutes without gel electrophoresis. All the CP-CRE isolates (100%) were positive for either NDM or OXA-48 by PCR-dipstick DNA Chromatography assay, whereas 142 (97.9%) isolates were detected by conventional PCR. The positivity rate of NDM, OXA-48 and both NDM and OXA-48 among CP-CRE isolates were 73.8%, 51.7% and 25.5% respectively. The sensitivity, specificity of PCR-dipstick DNA Chromatography assay for detection of NDM and OXA-48 were 100.0%, 92.7% and 98.6%, 97.2% respectively. This assay showed excellent agreement with conventional PCR for detection of CP-CRE. PCR-dipstick DNA chromatography may be an excellent and robust diagnostic tool in terms of rapidity and sensitivity for the detection of CP-CRE.

Detection of breast cancer-related point-mutations using screen-printed and gold-plated electrochemical sensor arrays suitable for point-of-care applications

For anticancer therapy and disease prognosis in breast cancer, three PIK3CA point-mutations (H1047R, E545K, and E542K) play a significant role. To allow for specific and sensitive detection of these point-mutations with a hybridization-based detection concept, the assay conditions were optimized on a microarray technology platform. The resulting fluorescence-based microarray assay enables simultaneous and specific detection of three PIK3CA point-mutations. The optimized protocol was then adapted for use on a screen-printed and gold-plated silver sensor array with twelve working electrodes, one common counter electrode, and one common reference electrode. Chronoamperometric measurements employing an enzyme-amplified electrochemical assay allow for detecting PIK3CA point-mutations with a detection limit of 10 pM for short 24-mer target DNA. The mutant and the wild-type target DNA sequences gave significantly different signals in a broad concentration range of 1 nM – 100 nM, with the best separation found at 10 nM – 20 nM. Comparing the hybridization of short 24-mer and long 80-mer target DNA sequences reveals that the hybridization efficiency is reduced for long target DNA sequences. However, both the 24-mer and the 80-mer target DNA lead to successful detection of point-mutations. Finally, the electrochemical sensor allows for multiplexed detection of the three PIK3CA point-mutations.

Depletion of wild-type target enhances the hybridization-based sensitivity of low-abundant mutation detection by reference capture probes

Nucleic acids duplex formation via hybridization is a crucial reaction in many processes and application across different disciplines. In life sciences the detection of mutations is an important application for which hybridization is used, e.g. in diagnostics via single-nucleotide variants (SNVs). This paper deals with the physico-chemical aspects of hybridization-based detection of low-abundance mutations, which is challenging due to unavoidable competitive hybridization of high-abundant wild type sequence with the low-abundant variants. We apply two experimental methods based on theoretical hybridization models to show how sensing of DNA mutation can be significantly improved. This is implemented on two SNV biomarkers for which we first select a reference capture probe. This is a probe designed to match neither the wild type nor the SNV sequence, but to have an equal affinity to the wild-type as the SNV-matching probe. This allows the mutation-specific signal to be expressed as a ratiometric quantity, leading to increased assay robustness. Secondly, we selectively deplete the wild-type species by introducing an excess of wild-type-specific capture probes, and account for these depletion effects in the theoretical model. We demonstrate the detection of 0.05% mutant species in a wild-type background, which is an improvement of an order of magnitude in the limit of detection in comparison with the no-depletion case. This sensitivity is comparable with digital PCR results, showing performance suitable for e.g. clinical applications in liquid biopsy context. The principles of this work apply to a wide range of hybridization-based DNA biosensing technologies, irrespective of the underlying transducer principle.

Single-Molecule Sensor for High-Confidence Detection of miRNA.

MicroRNAs (miRNAs) play a crucial role in regulating gene expression and have been linked to many diseases. Therefore, sensitive and accurate detection of disease-linked miRNAs is vital to the emerging revolution in early diagnosis of diseases. While the detection of miRNAs is a challenge due to their intrinsic properties such as small size, high sequence similarity among miRNAs and low abundance in biological fluids, the majority of miRNA-detection strategies involve either target/signal amplification or involve complex sensing designs. In this study, we have developed and tested a DNA-based fluorescence resonance energy transfer (FRET) sensor that enables ultrasensitive detection of a miRNA biomarker (miRNA-342-3p) expressed by triple-negative breast cancer (TNBC) cells. The sensor shows a relatively low FRET state in the absence of a target but it undergoes continuous FRET transitions between low- and high-FRET states in the presence of the target. The sensor is highly specific, has a detection limit down to low femtomolar (fM) without having to amplify the target, and has a large dynamic range (3 orders of magnitude) extending to 300 000 fM. Using this strategy, we demonstrated that the sensor allows detection of miRNA-342-3p in the miRNA-extracts from cancer cell lines and TNBC patient-derived xenografts. Given the simple-to-design hybridization-based detection, the sensing platform developed here can be used to detect a wide range of miRNAs enabling early diagnosis and screening of other genetic disorders.

Single‐molecule, hybridization‐based strategies for short nucleic acids detection and recognition with nanopores

DNA nanotechnology has seen large developments over the last 30 years through the combination of detection and discovery of DNAs, and solid phase synthesis to increase the chemical functionalities on nucleic acids, leading to the emergence of novel and sophisticated in features, nucleic acids-based biopolymers. Arguably, nanopores developed for fast and direct detection of a large variety of molecules, are part of a revolutionary technological evolution which led to cheaper, smaller and considerably easier to use devices enabling DNA detection and sequencing at the single-molecule level. Through their versatility, the nanopore-based tools proved useful biomedicine, nanoscale chemistry, biology and physics, as well as other disciplines spanning materials science to ecology and anthropology. This mini-review discusses the progress of nanopore- and hybridization-based DNA detection, and explores a range of state-of-the-art applications afforded through the combination of certain synthetically-derived polymers mimicking nucleic acids and nanopores, for the single-molecule biophysics on short DNA structures.

Microheater-integrated zinc oxide nanowire microfluidic device for hybridization-based detection of target single-stranded DNA

Detection of cell-free DNA (cfDNA) has an impact on DNA analysis in liquid biopsies. However, current strategies to detect cfDNA have limitations that should be overcome, such as having low sensitivity and requiring much time and a specialized instrument. Thus, non-invasive and rapid detection tools are needed for disease prevention and early-stage treatment. Here we developed a device having a microheater integrated with zinc oxide nanowires (microheater-ZnO-NWs) to detect target single-stranded DNAs (ssDNAs) based on DNA probe hybridization. We confirmed experimentally that our device realized in-situ annealed DNA probes by which we subsequently detected target ssDNAs. We envision that this device can be utilized for fundamental studies related to nanobiodevice-based DNA detection.

Single-Molecule FRET-Based Dynamic DNA Sensor.

Selective and sensitive detection of nucleic acid biomarkers is of great significance in early-stage diagnosis and targeted therapy. Therefore, the development of diagnostic methods capable of detecting diseases at the molecular level in biological fluids is vital to the emerging revolution in the early diagnosis of diseases. However, the vast majority of the currently available ultrasensitive detection strategies involve either target/signal amplification or involve complex designs. Here, using a p53 tumor suppressor gene whose mutation has been implicated in more than 50% of human cancers, we show a background-free ultrasensitive detection of this gene on a simple platform. The sensor exhibits a relatively static mid-FRET state in the absence of a target that can be attributed to the time-averaged fluorescence intensity of fast transitions among multiple states, but it undergoes continuous dynamic switching between a low- and a high-FRET state in the presence of a target, allowing a high-confidence detection. In addition to its simple design, the sensor has a detection limit down to low femtomolar (fM) concentration without the need for target amplification. We also show that this sensor is highly effective in discriminating against single-nucleotide polymorphisms (SNPs). Given the generic hybridization-based detection platform, the sensing strategy developed here can be used to detect a wide range of nucleic acid sequences enabling early diagnosis of diseases and screening genetic disorders.

Electrochemical biosensor for miRNA-21 based on gold-platinum bimetallic nanoparticles coated 3-aminopropyltriethoxy silane

We report an electrochemical biosensor based on gold platinum bimetallic nanoparticles (AuPtBNPs)/3-aminopropyltriethoxy silane (APTS) nanocomposite coated fluorine-doped tin oxide (FTO) as a biosensing platform for hybridization-based detection of miRNA-21. Field Emission-Scanning Electron Microscopy (FE-SEM), Fourier Transform Infrared Spectroscopy (FT-IR) and electrochemical measurements were carried out to ensure the successful construction of the biosensor. The amount of cDNA immobilized on electrode surface and hybridization time required for the miRNA-21 sensing were optimized. The biosensing platform showed detection limit of 0.63 fM with wide linear range i.e. 1 fM-100 nM for miRNA-21 detection. The biosensing strategy demonstrates a good recovery yield from 90.18% to 94.6% in serum samples. It offers good selectivity for its complementary miRNA compared to the non-complementary miRNAs. Other analytical features of the biosensor such as stability, reusability and reproducibility were also tested, providing appropriate results.

Dual-Signal Readout of DNA Methylation Status Based on the Assembly of a Supersandwich Electrochemical Biosensor without Enzymatic Reaction.

A highly sensitive and selective biosensing system was designed to analyze DNA methylation using a dual-signal readout technique in combination with the signal amplification of supersandwich DNA structure. Through the ingenious design of target-triggered cascade of hybridization chain reaction, one target DNA could initiate the formation of supersandwich structure with multiple signal probes. As a result, one-to-multiple amplification effect was achieved, which conferred high sensitivity to target molecular recognition. Based on probe 1 labeled with ferrocene and probe 2 modified with methylene blue, the target DNA was clearly recognized by two electrochemical signals at independent potentials, which was helpful for the acquisition of more accurate detection results. Taking advantage of bisulfite conversion, the methylation status of cytosine (C) was changed to nucleic acid sequence status, which facilitated the hybridization-based detection without enzymatic reaction. Consequently, the methylated DNA was detected at the femtomolar level with satisfactory analytical parameters. The proposed system was effectively used to assess methylated DNA in human blood serum samples, illuminating the possibility of the sensing platform for applications in disease diagnosis and biochemistry research.

DNA Adduct-Directed Synthetic Nucleosides.

Chemical damage to DNA is a key initiator of adverse biological consequences due to disruption of the faithful reading of the genetic code. For example, O6-alkylguanine ( O6-alkylG) DNA adducts are strongly miscoding during DNA replication when the damaged nucleobase is a template for polymerase-mediated translesion DNA synthesis. Thus, mutations derived from O6-alkylG adducts can have severe adverse effects on protein translation and function and are an early event in the initiation of carcinogenesis. However, the low abundance of these adducts places significant limitations on our ability to relate their presence and biological influences with resultant mutations or disease risk. As a consequence, there is a critical need for novel tools to detect and study the biological role of alkylation adducts. Incorporating DNA bases with altered structures that are derived synthetically is a strategy that has been used widely to interrogate biological processes involving DNA. Such synthetic nucleosides have contributed to our understanding of DNA structure, DNA polymerase (Pol) and repair enzyme function, and to the expansion of the genetic alphabet. This Account describes our efforts toward creating and applying synthetic nucleosides directed at DNA adducts. We synthesized a variety of nucleosides with altered base structures that complement the altered hydrogen bonding capacity and hydrophilicity of O6-alkylG adducts. The heterocyclic perimidinone-derived nucleoside Per was the first of such adduct-directed synthetic nucleosides; it specifically stabilized O6-benzylguanine ( O6-BnG) in a DNA duplex. Structural variants of Per were used to determine hydrogen bonding and base-stacking contributions to DNA duplex stability in templates containing O6-BnG as well as O6-methylguanine ( O6-MeG) adducts. We created synthetic probes able to stabilize damaged over undamaged templates and established how altered hydrogen bonding or base-stacking properties impact DNA duplex stability as a function of adduct structures. This knowledge was then applied to devise a hybridization-based detection strategy involving gold nanoparticles that distinguish damaged from undamaged DNA by colorimetric changes. Furthermore, synthetic nucleosides were used as mechanistic tools to understand chemical determinants such as hydrogen bonding, π-stacking, and size and shape deviations that impact the efficiency and fidelity of DNA adduct bypass by DNA Pols. Finally, we reported the first example of amplifying alkylated DNA, accomplished by combining an engineered polymerase and synthetic triphosphate for which incorporation is templated by a DNA adduct. The presence of the synthetic nucleoside in amplicons could serve as a marker for the presence and location of DNA damage at low levels in DNA strands. Adduct-directed synthetic nucleosides have opened new concepts to interrogate the levels, locations, and biological influences of DNA alkylation.

Optimizing peptide nucleic acid probes for hybridization-based detection and identification of bacterial pathogens.

Point-of-care (POC) diagnostics for infectious diseases have the potential to improve patient care and antibiotic stewardship. Nucleic acid hybridization is at the core of many amplification-free molecular diagnostics and detection probe configuration is key to diagnostic performance. Modified nucleic acids such as peptide nucleic acid (PNA) offer advantages compared to conventional DNA probes allowing for faster hybridization, better stability and minimal sample preparation for direct detection of pathogens. Probes with tethered fluorophore and quencher allow for solution-based assays and eliminate the need for washing steps thereby facilitating integration into microfluidic devices. Here, we compared the sensitivity and specificity of double stranded PNA probes (dsPNA) and PNA molecular beacons targeting E. coli and P. aeruginosa for direct detection of bacterial pathogens. In bulk fluid assays, the dsPNAs had an overall higher fluorescent signal and better sensitivity and specificity than the PNA beacons for pathogen detection. We further designed and tested an expanded panel of dsPNA probes for detection of a wide variety of pathogenic bacteria including probes for universal detection of eubacteria, Enterobacteriaceae family, and P. mirablis. To confirm that the advantage translated to other assay types we compared the PNA beacon and dsPNA in a prototype droplet microfluidic device. Beyond the bulk fluid assay and droplet devices, use of dsPNA probes may be advantageous in a wide variety of assays that employ homogenous nucleic acid hybridization.

Large-scale validation of miRNAs by disease association, evolutionary conservation and pathway activity

ABSTRACTThe validation of microRNAs (miRNAs) identified by next generation sequencing involves amplification-free and hybridization-based detection of transcripts as criteria for confirming valid miRNAs. Since respective validation is frequently not performed, miRNA repositories likely still contain a substantial fraction of false positive candidates while true miRNAs are not stored in the repositories yet. Especially if downstream analyses are performed with these candidates (e.g. target or pathway prediction), the results may be misleading. In the present study, we evaluated 558 mature miRNAs from miRBase and 1,709 miRNA candidates from next generation sequencing experiments by amplification-free hybridization and investigated their distributions in patients with various disease conditions. Notably, the most significant miRNAs in diseases are often not contained in the miRBase. However, these candidates are evolutionary highly conserved. From the expression patterns, target gene and pathway analyses and evolutionary conservation analyses, we were able to shed light on the complexity of miRNAs in humans. Our data also highlight that a more thorough validation of miRNAs identified by next generation sequencing is required. The results are available in miRCarta (https://mircarta.cs.uni-saarland.de).

PCR-Dipstick Chromatography for Differential Detection of Carbapenemase Genes Directly in Stool Specimens.

A PCR-dipstick chromatography technique was designed and evaluated for differential identification of blaNDM, blaKPC, blaIMP, and blaOXA-48 carbapenemase genes directly in stool specimens within 2 h. It is a DNA-DNA hybridization-based detection system where PCR products can be easily interpreted by visual observation without electrophoresis. The PCR-dipstick showed high sensitivity (93.3%) and specificity (99.1%) in directly detecting carbapenemase genes in stool specimens compared with multiplex PCR for genomic DNA of the isolates from those stool specimens.

Highly multiplex and sensitive SNP genotyping method using a three-color fluorescence-labeled ligase detection reaction coupled with conformation-sensitive CE.

For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost-effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation-dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error-prone hybridization-based detection, the multiplex assay process is complicated because of the size-based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high-resolution CE-SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar-sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three-color fluorescence-labeled probes.

Direct detection and drug-resistance profiling of bacteremias using inertial microfluidics.

Detection of bacteria in bloodstream infections and their antibiotic susceptibility patterns is critical to guide therapeutic decision-making for optimal patient care. Current culture-based assays are too slow (>48 h), leading to excessive up-front use of broad-spectrum antibiotics and/or incorrect antibiotic choices due to resistant bacteria, each with deleterious consequences for patient care and public health. To approach this problem, we describe a method to rapidly isolate bacteria from whole blood using inertial microfluidics and directly determine pathogen identity and antibiotic susceptibility with hybridization-based RNA detection. Using the principle of Dean flow fractionation, bacteria are separated from host blood cells in a label-free separation method with efficient recovery of even low abundance bacteria. Ribosomal RNA detection can then be applied for direct identification of low abundance pathogens (~100 per mL) from blood without culturing or enzymatic amplification. Messenger RNA detection of antibiotic-responsive transcripts after brief drug exposure permits rapid susceptibility determination from bacteria with minimal culturing (~10(5) per mL). This unique coupling of microfluidic cell separation with RNA-based molecular detection techniques represents significant progress towards faster diagnostics (~8 hours) to guide antibiotic therapy.

An integrated closed-tube 2-plex PCR amplification and hybridization assay with switchable lanthanide luminescence based spatial detection.

Switchable lanthanide luminescence is a binary probe technology that inherently enables a high signal modulation in separation-free detection of DNA targets. A luminescent lanthanide complex is formed only when the two probes hybridize adjacently to their target DNA. We have now further adapted this technology for the first time in the integration of a 2-plex polymerase chain reaction (PCR) amplification and hybridization-based solid-phase detection of the amplification products of the Staphylococcus aureus gyrB gene and an internal amplification control (IAC). The assay was performed in a sealed polypropylene PCR chip containing a flat-bottom reaction chamber with two immobilized capture probe spots. The surface of the reaction chamber was functionalized with NHS-PEG-azide and alkyne-modified capture probes for each amplicon, labeled with a light harvesting antenna ligand, and covalently attached as spots to the azide-modified reaction chamber using a copper(i)-catalyzed azide-alkyne cycloaddition. Asymmetric duplex-PCR was then performed with no template, one template or both templates present and with a europium ion carrier chelate labeled probe for each amplicon in the reaction. After amplification europium fluorescence was measured by scanning the reaction chamber as a 10 × 10 raster with 0.6 mm resolution in time-resolved mode. With this assay we were able to co-amplify and detect the amplification products of the gyrB target from 100, 1000 and 10,000 copies of isolated S. aureus DNA together with the amplification products from the initial 5000 copies of the synthetic IAC template in the same sealed reaction chamber. The addition of 10,000 copies of isolated non-target Escherichia coli DNA in the same reaction with 5000 copies of the synthetic IAC template did not interfere with the amplification or detection of the IAC. The dynamic range of the assay for the synthetic S. aureus gyrB target was three orders of magnitude and the limit of detection of 8 pM was obtained. This proof-of-concept study shows that the switchable lanthanide luminescent probes enable separation-free array-based multiplexed detection of the amplification products in a closed-tube PCR which can enable a higher degree of multiplexing than is currently feasible by using different spectrally separated fluorescent probes.

Tree-side Molecular Testing for DNA from the HLB Bacterium Candidatus Liberibacter asiaticus

HLB affects all citrus cultivars and causes tree decline resulting in greatly reduced citrus production in Asia, Africa, the Indian subcontinent, the Arabian Peninsula, Brazil, and the United States. To enable integrated pest management strategies for combating HLB, we have developed a sensitive nucleic acid-based HLB diagnostic test based on Mesa Tech International, Inc.’s (MTI's) new point-of-use diagnostic platform, MTIDx, and targeting hyvI/hyvII genes of Candidatus Liberibacter asiaticus (Las) (Zhou et al., 2011). The MTIDx platform integrates sample preparation, rapid nucleic acid amplification and sequence-specific hybridization-based detection. The simplicity of the test may offer end users with little or no specialized training the opportunity to obtain molecular test results comparable to laboratory-based PCR methods without costly instrumentation. Here, we will report on the efficacy of MTI's nucleic acid testing platform for the detection of Candidatus Liberibacter asiaticus bacteria in greenhouse and field collected citrus tissue and in the insect vector of the pathogen (Diaphorina citri Kuwayama). Comparative studies evaluating the MTIDx platform's performance relative to widely accepted laboratory PCR testing will be presented. Results from these studies will be discussed in the context of their implications for HLB management.