Isothermal Reaction Conditions Research Articles

Ultra-sensitive colorimetric detection of SARS-CoV-2 by novel gold nanoparticle (AuNP)-assisted loop-mediated isothermal amplification (LAMP) and freezing methods.

Loop-mediated isothermal amplification (LAMP) is a molecular diagnosis technology with the advantages of isothermal reaction conditions and high sensitivity. However, the LAMP reactions are prone to producing false-positive results and thus are usually less reliable. This study demonstrates a gold nanoparticle (AuNP)-assisted colorimetric LAMP technique for diagnosing SARS-CoV-2, which aims to overcome the false-positive results. The AuNPs were functionalized with E gene probes, specifically tailored to bind to the amplified E-gene LAMP product, using the freezing method. Varied salt concentration and AuNP/probe combinations were tested for the highest visual performance. The experiments were conducted on synthetic SARS-CoV-2 RNA (Omicron variant), as well as on clinical samples. The assay showed an exceptional sensitivity of 8.05fg of LAMP amplicon mixture (0.537fg/µL). The average reaction time was ~ 30min. In conclusion, AuNP-assisted LAMP detection will not identify any potential unspecific amplification, which helps to improve the efficiency and reliability of LAMP assays in point-of-care applications. The freezing method to functionalize the AuNPs with probes simplifies the assay, which can be utilized in further diagnostic studies.

Syngas Production by Chemical Looping Dry Reforming of Methane over Ni-modified MoO3/ZrO2.

We investigated supported-MoO3 materials effective for the chemical looping dry reforming of methane (CL-DRM) to decrease the reaction temperature. Ni-modified molybdenum zirconia (Ni/MoO3/ZrO2) showed CL-DRM activity under isothermal reaction conditions of 650 °C, which was 100-200 °C lower than the previously reported oxide-based materials. Ni/MoO3/ZrO2 activity strongly depends on the MoO3 loading amount. The optimal loading amount was 9.0 wt.% (Ni/MoO3(9.0)/ZrO2), wherein two-dimensional polymolybdate species were dominantly formed. Increasing the loading amount to more than 12.0 wt.% resulted in a loss of activity owing to the formation of bulk Zr(MoO4)2 and/or MoO3. In situ Mo K-edge XANES studies revealed that the surface polymolybdate species serve as oxygen storage sites. The Mo6+ species were reduced to Mo4+ species by CH4 to produce CO and H2. The reduced Mo species reoxidized by CO2 with the concomitant formation of CO. The developed Ni/MoO3(9.0)/ZrO2 was applied to the long-term CL-DRM under high concentration conditions (20 % CH4 and 20 % CO2) at 650 °C, with two pathways possible for converting CH4 and CO2 to CO and H2 via the redox reaction of the Mo species and coke formation.

Same size, same support, same spectator? Selective acetylene hydrogenation on supported Pd nanoparticles.

The selective hydrogenation of acetylene catalyzed by Pd nanoparticles is industrially used to increase the purity of ethylene. Despite the implementation of Pd based catalysts on an industrial scale, little is known about metal-support interactions on a fundamental level due to the complexity of these systems. In this study, the influence of metal-support interactions between Pd nanoparticles and two electronically modified a-SiO2 thin films on acetylene hydrogenation is investigated under ultra-high vacuum (UHV) conditions. The hydrogenation is performed under isothermal reaction conditions using a pulsed molecular beam reactive scattering (pMBRS) technique. Besides the activity and selectivity of clean Pd particles also the impact of dehydrogenated species intentionally introduced a priori is elucidated, whereas the active phase of the catalyst is additionally characterized by CO infrared reflection-absorption spectroscopy (IRRAS) and post-mortem temperature-programmed reaction (TPR). Metal-support interactions are found to influence the catalytic properties of Pd particles by charge-transfer, where positive charging leads to increased activity for acetylene hydrogenation. However, the increased activity is accompanied by formation of undesired byproducts. The active sites for acetylene and ethylene hydrogenation are shown to be different as previously proposed by the A and E model. The availability of the two different active sites on the Pd nanoparticles is determined by dehydrogenated species, whose nature and stability can be tuned by metal-support interactions. Based on these findings an electronic model is proposed how selectivity for acetylene hydrogenation can be steered solely by metal-support interactions leading to blocking of unselective sites in situ.

Oxidative coupling of biogas to ethylene over a trilobe-shaped Mn-Na2WO4/α-Al2O3 catalyst in a single-pellet reactor

Despite the development of selective catalyst formulations for oxidative coupling of methane (OCM), kinetic and reactor design studies have been hampered by a lack of data for truly isothermal reaction conditions. Because of the high exothermicity, the temperature of a fixed-bed reactor filled with Mn-Na2WO4/SiO2 catalysts will show hot-spots with a loss of selectivity. The objective of this study is to measure catalyst activity under relevant reaction conditions of pressure (3 bar), biogas composition (no dilution) and contact time, while keeping the process isothermal. To this aim, we have synthesized and shaped a trilobed Mn-Na2WO4/α-Al2O3 catalyst, which may be used as is at industrial scale, and measured its performance in a single-pellet reactor. We found that the presence of CO2 did not degrade the catalyst performance, even on the long run, which should enable the use of a CO2-rich stream such as biogas. Actually, higher methane conversions have been obtained in the presence of CO2, likely due to a possible contribution of methane dry reforming reaction. We also noticed a slow catalyst activation over the first 100 h, while selectivity was kept constant.

Investigating the mechanochemistry of membrane area asymmetry generation: A combined mass-action and micromechanical study using methyl-beta-cyclodextrin.

Membrane asymmetry is important for cell signaling and is coupled with membrane morphological remodeling protein conformational regulation, sorting, and function. With respect to the lipids composing a bilayer, membrane asymmetry is divided into two contributions including 1) area asymmetry- that is, unequal monolayer surface areas that two apposed leaflets would tend to favor were they uncoupled and 2) compositional asymmetry- that is, different lipid compositions between the leaflets. For in vitro research on membrane asymmetry, methyl-β-cyclodextrin (mbCD) has been previously applied as a lipid-exchange catalyst to tune the compositional asymmetry of synthetic membranes. Inspired by this approach, we opted to study the physical chemistry of 1-component vesicle systems where mbCD was present not as a catalyst but as a reactant. In reaction mixtures at ambient conditions containing nanoscale vesicles, we performed a fluoresence-quenching assay to see that mbCD extracts phospholipids from only the outer leaflets of vesicles and, thereby, must generate area asymmetry in such mixtures. For certain molar ratios, mbCD can fully solubilize all phospholipid and we used 90o light scattering to map this behavior on a two-dimensional phase diagram at ambient temperature. Through temperature-cycling experiments, we showed an irreversibility in mbCD-phospholipid phase behavior. This granted access to stable solutions containing no self-assembled lipid structures with larger molar ratios of phospholipid to mbCD than could be achieved by isothermal reaction conditions. By adjusting the molar ratios of these solutions, we generated both negative and positive curvature in microscopic vesicles (i.e., inward and outward bending, respectively). Tunability of the curvature is consistent with mass-action considerations relating to net flow of phospholipid out of/into vesicles. We believe our approach using mbCD pre-complexed with phospholipid will enhance experimental research on the biophysical consequences of area asymmetry.

Multiple ligation–Assisted recombinase polymerase amplification for highly sensitive and selective colorimetric detection of SARS-CoV-2

In this paper we present a new method for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), targeting a specific region “N gene.” Under isothermal reaction conditions, we integrated ligation (Lig; high selectivity) and recombinase polymerase amplification (RPA; high sensitivity) processes, obtaining a robust method of detection. For point-of-care testing, we incorporated our laboratory-produced pyrophosphate ion (PPi)–sensing probe (PK-probe) for colorimetric analysis of the reaction. The total detection system was efficient and effective at diagnosing this RNA virus–mediated disease rapidly (30 min). In a full-genome SARS-CoV-2 study, our PK-probe/Lig-RPA system functioned with a limit of detection of 1160 copies/ml, with a single-mismatch level of selectively, and it was highly selective even in the presence of bacterial genomes commonly found in the human mouth and nose. This robust, straightforward, selective, efficient, and ultrasensitive colorimetric detection method, with potential for point-of-care analysis, should also be effective in detecting a diverse range of other RNA-based diseases.

Highly Specific Loop-Mediated Isothermal Amplification Using Graphene Oxide-Gold Nanoparticles Nanocomposite for Foot-and-Mouth Disease Virus Detection.

Loop-mediated isothermal amplification (LAMP) is a molecular diagnosis technology with the advantages of rapid results, isothermal reaction conditions, and high sensitivity. However, this diagnostic system often produces false positive results due to a high rate of non-specific reactions caused by formation of hairpin structures, self-dimers, and mismatched hybridization. The non-specific signals can be due to primers used in the methods because the utilization of multiple LAMP primers increases the possibility of self-annealing of primers or mismatches between primers and templates. In this study, we report a nanomaterial-assisted LAMP method that uses a graphene oxide–gold nanoparticles (AuNPs@GO) nanocomposite to enable the detection of foot-and-mouth disease virus (FMDV) with high sensitivity and specificity. Foot-and-mouth disease (FMD) is a highly contagious and deadly disease in cloven-hoofed animals; hence, a rapid, sensitive, and specific detection method is necessary. The proposed approach exhibited high sensitivity and successful reduction of non-specific signals compared to the traditionally established LAMP assays. Additionally, a mechanism study revealed that these results arose from the adsorption of single-stranded DNA on AuNPs@GO nanocomposite. Thus, AuNPs@GO nanocomposite is demonstrated to be a promising additive in the LAMP system to achieve highly sensitive and specific detection of diverse diseases, including FMD.

Partial oxidation of methanol to formaldehyde in an annular reactor

A structured reactor with annular configuration was applied for studying methanol oxidation to formaldehyde over silver. By eliminating gas phase reactions, high formaldehyde selectivity (93–97%) was obtained at low methanol and oxygen conversion under practically isothermal reaction conditions. CH2O and CO2 were the only carbon containing products, and both may be claimed as primary products along with H2. It also proves that CO is formed by homogenous decomposition of CH2O and should not be considered a main precursor to CO2, as assumed in several reaction mechanisms. The analysis of H2/CO2 ratio as a function of temperature provides an estimate of contributions from dehydrogenation and partial oxidation of methanol, and clearly suggests presence of a dehydrogenation pathway to CH2O. Extracting kinetic parameters is challenging due to a correlation between activity, oxygen dissolution, and silver restructuring and morphology and its dependence on temperature. Nevertheless, the data indicate 1st order with respect to oxygen. Conditioning by reaction at high temperature followed by a temperature ramp was performed to minimize the impact of a gradually changing Ag catalyst. The resulting Arrhenius analysis implies two distinct regions of activity. The apparent activation energy was estimated to 41 kJ/mol for the high temperature region, a value close to the activation energy for oxygen diffusion in silver at high temperature. The investigation demonstrates benefits of using an annular reactor configuration in bridging lab scale investigations with industrial conditions. Collecting reaction data at low oxygen conversion is enabled, which has not been achievable in conventional lab scale reactors this far.

Isothermal redox cycling of A‐ and B‐site substituted manganite‐based perovskites for CO2 conversion

AbstractConversion of CO2 into fuels and valuable chemicals can simultaneously address two major global issues: climate change and the increasing energy demand. Most of the previous studies regarding the thermochemical splitting of CO2 into CO have been carried out at elevated temperatures of up to 1400°C. In this study, under isothermal reaction conditions, manganite‐based perovskites are used to convert CO2 to CO in a thermogravimetric analyzer (TGA). In addition, the use of CH4 as a reducing agent has lowered the reaction temperatures (≤900°C). This study primarily focuses on the influence of A‐ and B‐site substitution on the redox performances of the manganite‐based perovskites. Based on the redox performances of four samples (CaMnO3, La0.5Ca0.5MnO3, La0.5Sr0.5MnO3, and Y0.5Sr0.5MnO3) with different A‐site compositions, La0.5Sr0.5MnO3 displays the best stability. Similarly, in the La1‐xSrxMnO3 perovskite family (x = 0.5, 0.25, and 0.10), the performance of La0.5Sr0.5MnO3 outshines the other two compositions. It is observed that as the Sr content increases, the activation energy for reduction decreases. Substituting Mn with Al and Fe in the B‐site of the La1‐xSrxMnO3 perovskite, did not enhance the performance. The perovskite materials investigated have shown decrease in activity after the first cycle, possibly due to sintering and powder densification. However, after the first cycle, the perovskites illustrated good stability for 10 redox cycles. In this paper, size variance and metal‐oxygen bond strength provide the best explanation for the trends observed during the reduction of the perovskites.

Universal and Naked-Eye Gene Detection Platform Based on the Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a/13a System

Gold-nanoparticles-based colorimetric assay is an attractive detection format, but is limited by the tedious and ineffective posthybridization manipulations for genomic analysis. Here, we present a new design for a colorimetric gene-sensing platform based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. In this strategy, programmable recognition of DNA by Cas12a/crRNA and RNA by Cas13a/crRNA with a complementary target activates the trans-ssDNA or -ssRNA cleavage. Target-induced trans-ssDNA or ssRNA cleavage triggers an aggregation behavior change for the designed AuNPs-DNA probes pair, enabling the completion of naked-eye gene detection (transgenic rice, African swine fever virus, and miRNAs as the models) within 1 h. This platform is also showing promise as a fast and inexpensive tool for bacteria identification using 16S rDNA or 16S rRNA. A CRISPR/Cas-based colorimetric platform shows superior characteristics, such as probe universality, compatibility with isothermal reaction conditions, on-site detection capability, and high sensitivity, thus, demonstrating its use as a robust next-generation gene detection platform.

Effect of Cyclic Reaction on Corrosion Behavior of Chromium-Containing Alloys in CO2 Gas at 650 °C

In this work, seven commercial alloys (602CA, 310SS, 253MA, F321, F316L, 800H and 304SS) were investigated in Ar–20% CO2 gas at 650 °C under a cyclic condition (1-h reaction and 0.25-h cooling in each cycle) for up to 310 cycles. The corrosion behavior of these alloys in isothermal reaction condition was also carried out for a purpose of comparison. The results showed that nickel-based 602CA alloy stayed protective in both isothermal and cyclic reaction conditions by forming a thin protective alumina scale. However, alloys F321, F316L, 800H and 304SS all formed thick multilayered oxides with external iron-rich oxides and internal spinel oxides in all reaction conditions. Alloys 310SS and 253MA behaved protective in isothermal reaction condition but formed pitting corrosion in cyclic reaction condition. The high corrosion resistance of 310SS and 253MA was attributed to the high Cr content and the effect of other alloying elements, e.g., Si and Mn, forming additional oxide layers to enhance chromia protection. Cyclic reaction created stress on oxide scale during cooling and heating which accelerated the initiation of breakaway corrosion of these alloys. Carburization due to CO2 reaction was identified for F321, F316L and 304SS, but not for other alloys because of the formation of highly protective alumina or chromia scales.

Diffusion- and Mobility-Controlled Self-Healing Polymer Networks with Dynamic Covalent Bonding

A systematic study of diffusion-controlled reversible Diels–Alder (DA) network formation is performed under both isothermal and nonisothermal reaction conditions based on two amorphous furan–maleim...

Integration of rolling circle amplification and optomagnetic detection on a polymer chip

Rolling circle amplification (RCA) combined with padlock probe recognition of a DNA target is attractive for on-chip nucleic acid testing due to its high specificity and isothermal reaction conditions. However, the integration of RCA on an automated chip platform is challenging due to the different reagents needed for the reaction steps and the temperature sensitivity of the phi29 polymerase. Here, we describe the integration of an RCA assay on a single-use polymer chip platform where magnetic microbeads are used as solid support to transport the DNA target between three connected reaction chambers for (i) padlock probe annealing and ligation, (ii) RCA, and (iii) optomagnetic detection of RCA products. The three chambers were loaded with reagents by sequential filling combined with passive microfluidic structures. After loading, the on-chip assay steps were automated. For an assay in which all steps but the padlock probe annealing on the target were performed on-chip, we found a limit of detection (LOD) for a synthetic influenza target of 2 pM after 45 min of RCA, which is comparable to the corresponding laboratory assay. The entire assay, including padlock probe annealing, could be performed on-chip with an LOD of 20 pM after 45 min of RCA. This LOD can likely be reduced by further optimizing the microbead mixing. The results present important steps towards the integration and automation of RCA and potentially also other complex multi-step assays on a single-use polymer chip for molecular analysis.

Subzymes: Regulating DNAzymes for point of care nucleic acid sensing

Rapid, sensitive and affordable nucleic acid sensors that can operate in a point of care (POC) setting are highly desired; however, their availability and implementation remain limited. We report the development of a strategy for building sensors which use novel catalytic nucleic acid structures, herein referred to as ‘Subzymes’. We examine the effectiveness of Subzymes to mediate signal amplification in a universal manner allowing faster rates of detection. We created Subzymes by manufacturing composite oligonucleotides that contain a catalytic nucleic acid component and a substrate component. We demonstrate that the activity of some catalytic DNAzyme components can be inhibited by attaching the Subzymes to micro-particles. Subsequent cleavage of a Subzyme’s internal substrate results in the release and activation of the surface-bound DNAzyme, thus providing a mechanism to control the catalytic activity of the DNAzyme. We demonstrate that released DNAzymes are capable of cleaving fluorescent-labelled reporter substrates to generate a signal, thus confirming the restoration of their catalytic activity. The addition of Subzymes to reactions where the detection of a target is achieved by multi-component nucleic acid enzymes, known as PlexZymes, showed improved target sensitivities in a shorter amount of time (10 pM of target detected in under 60 min), demonstrating rapid detection of nucleic acid targets without the use of protein-enzymes. Subzymes are constructed from low-cost materials and operate under isothermal reaction conditions which are advantageous for on-site diagnostic testing. Thus, Subzymes offer a universal, rapid and affordable tool for nucleic acid sensing, providing new avenues for POC testing.

Evaluation of the reverse transcription strand invasion based amplification (RT-SIBA) RSV assay, a rapid molecular assay for the detection of respiratory syncytial virus

Respiratory syncytial virus (RSV) causes acute respiratory infections. Rapid RSV diagnosis has an impact on patient management. In a newly developed molecular assay, named reverse transcription strand invasion based amplification (RT-SIBA) RSV assay, RSV RNA is reverse transcribed to cDNA and amplified and detected under isothermal reaction conditions. The performance of this assay was evaluated. Respiratory samples that tested positive (n = 81) or negative (n = 61) for RSV with the multiplex RT-PCR Anyplex II RV16 Detection Kit (Anyplex) were analyzed with the RT-SIBA assay. Discordant samples were tested with the GeneXpert Flu/RSV XC assay. Consistent results in at least 2 of the 3 methods were defined as reference standard. The RT-SIBA assay yielded a negative result for the 61 negative samples and a positive result in 71/81 (85.5%) of the Anyplex positive samples. After a resolution of discordant samples, the positive and negative percent agreement of the RT-SIBA assay were 92% and 100%, respectively. The RT-SIBA assay is a rapid molecular assay for the detection of RSV with good performance in clinical specimens.

Enzyme-free dual-amplification strategy for the rapid, single-step detection of nucleic acids based on hybridization chain reaction initiated entropy-driven circuit reaction

Developing robust, isothermal and simple nucleic acids detection strategies is of great significance to clinical diagnosis and bioanalysis. Recently, hybridization chain reaction has attracted intense interest owing to its isothermal reaction conditions, enzyme-free nature, and simple sequence design. However, strategies based on HCR alone may not offer satisfactory sensitivity when trace targets are required to be analyzed. Here, we developed an enzyme-free and isothermal dual-amplification sensing strategy for the rapid and one-step detection of nucleic acids based on hybridization chain reaction and entropy-driven circuit reaction (HCR-EDCR). The target DNA initiates upstream HCR to assemble polymeric double stranded DNA (dsDNA) nanowires composed of many tandem trigger units that motivate downstream EDCR to continuously liberate report strand, resulting in the generation of an amplified fluorescence readout signal. This isothermal and homogenous strategy exhibits high sensitivity and good selectivity with a limit of detection of 87 fM, without the involvement of any enzymes. The whole dual-amplification can be completed with one-step in 45 min. Moreover, the developed HCR-EDCR approach can be easily adapted for detecting different analytes just by substituting the target-specific sequence. Therefore, this cascaded strategy may offer a new promising model for various diagnostic and biological analysis.

Sensitive and specific detection of microRNAs based on two-stage amplification reaction using molecular beacons as turn-on probes

In this study, a rapid, sensitive, and specific assay for detecting miRNAs was developed based on a two-stage amplification reaction (TSAR) using molecular beacons (MBs) as turn-on probes. In the TSAR, different miRNAs can be converted to the same reporter oligonucleotides (Y), which can hybridize with the same MB. Therefore, in combination with specific templates, this method can be applied to multiplex miRNA detection by simply using the same MB. The loop region of the MB was screened by computer simulation methods. In particular, to improve the specificity of the MB in real sample analysis, the maximum similarity of the MB loop region to the human genome and human transcriptome is less than 70%. Two MBs were designed in this study. MB I, with nine flanking base pairs in its stem region, was used for real-time monitoring of the production of Y during the TSAR. MB II, with five flanking base pairs in its stem region, was used to detect the production of Y at the end of the TSAR. This assay exhibited high sensitivity with a limit of detection of 2.0 × 10−16M and 6.7 × 10−16M using MB I and MB II as turn-on probes, respectively. In addition, this assay can clearly discriminate single base differences in miRNA sequences, and the TSAR can be completed under isothermal conditions. Accordingly, the isothermal reaction conditions and simple fluorescence measurement can greatly contribute to the development of a fast point-of-care detection system.

Reverse transcription strand invasion based amplification (RT-SIBA): a method for rapid detection of influenza A and B.

Rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. Isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (PCR), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. We previously described a novel isothermal nucleic acid amplification method, ‘Strand Invasion Based Amplification’ (SIBA®), with high analytical sensitivity and specificity, for the detection of DNA. In this study, we describe the development of a variant of the SIBA method, namely, reverse transcription SIBA (RT-SIBA), for the rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows one-step reverse transcription of RNA to complementary DNA (cDNA) and simultaneous amplification and detection of the cDNA by SIBA under isothermal reaction conditions. The RT-SIBA method was found to be more sensitive than PCR for the detection of influenza A and B and could detect 100 copies of influenza RNA within 15 min. The development of RT-SIBA will enable rapid and accurate diagnosis of viral RNA targets within point-of-care or central laboratory settings.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7491-y) contains supplementary material, which is available to authorized users.

CO2 reforming of methane over Ni-Mg-Al-Ce mixed oxides derived from hydrotalcites: Mg/Ni ratio effect

Ni-Ce/Mg-Al catalysts were obtained by means of the hydrotalcite reconstruction method in the presence of [Ce(EDTA)]- complex. The effect of the Mg/Ni ratio was studied when the mixed oxide was reconstructed with 3 wt. % Ce loadings. The materials were characterized by X-Ray Diffraction (XRD), Thermal-Gravimetric Analysis (TGA), Temperature-Programmed Reduction (TPR-H2), Temperature-Programmed Oxidation (TPO) and CO2Temperature-Programmed Desorption (TPD-CO2). The reduced catalysts were tested in CO2 reforming of methane under two operation regimens: i) reaction between 500 to 800ºC using volumetric ratios of CH4/CO2/Ar=5/5/40 and, ii) isothermal reaction at 700ºC using volumetric ratios of CH4/CO2=18/22 without diluent gas. The solids showed strong basic characteristics and high Ni-surface interactions which determined their catalytic performance. Catalysts with Mg/Ni molar ratios of 2 and 4 showed high CH4 and CO2 conversions with H2/CO molar ratios between 0.7 and 1.1 without coke formation under severe isothermal reaction conditions. The yields and activities were higher when reduction increased.

Multiplex isothermal helicase-dependent amplification assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae

Thermophilic helicase dependent amplification (tHDA), which employs helicase to unwind double-stranded DNA at constant temperature, is a relatively new isothermal nucleic acid amplification technology. In this study, the development and optimization of a 4-plex tHDA assay for detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are described. tHDA is combined with sequence-specific sample preparation on magnetic beads and homogeneous endpoint fluorescence detection using dual-labeled probes. This 4-plex tHDA assay was applied to the detection of 2 genes on CT and a multicopy gene on NG in the presence of an internal control. The assay showed high analytical sensitivity and specificity of simultaneous CT/NG detection and is compatible with a wide variety of sample types and media. The isothermal reaction conditions and homogeneous endpoint detection utilized in this assay are well suited for laboratory automation and high-throughput screening applications as well as for point-of-care testing.