Synchronous detection of Burkholderia pseudomallei and its ceftazidime resistance mutation based on RNase-HII hydrolysis combined with lateral flow strip assay.

IMPORTANCE: This study focused on the development of a reaction system using rhPCR to amplify a specific gene, ORF2, of B. pseudomallei and to identify the P174L mutation associated with increased drug resistance to ceftazidime (CAZ). The system incorporated universal primer probes and a simple temperature cycle reaction. The amplified products were then analyzed using lateral flow strip assay (LFSA) for strain identification and mutation interpretation. The developed system provides a reliable basis for diagnosing melioidosis and selecting appropriate drugs. Its potential impact is particularly significant in resource-limited settings where access to advanced diagnostic techniques is limited. This platform stands out for its simplicity, convenience, sensitivity, specificity, and portability. It shows promise as a point-of-care testing method for detecting single nucleotide polymorphism in genes associated with other diseases. By leveraging the advantages of this platform, researchers and healthcare professionals can potentially expand its use beyond melioidosis and apply it to the rapid detection of genetic variations in other disease-related genes.

AuNPs@MoSe2 heterostructure as a highly efficient coreaction accelerator of electrocheluminescence for amplified immunosensing of DNA methylation.

Electrocheluminescence analysis amplified by coreaction accelerators has experienced breakthrough in ultrasensitive detection of biomarkers. Herein, a highly efficient coreaction accelerator, two-dimensional layered MoSe2 nanosheets loaded with gold nanoparticles (AuNPs@MoSe2 heterostructure), is proposed to enhance the ECL efficiency of Ru(bpy)32+/tripropylamine (TPrA) system. The presence of AuNPs avoids the aggregation of MoSe2 nanosheets, and improves the electrical conductivity of modified surface. The AuNPs@MoSe2 modified electrode also provides a large area for loading of abundant capture probe. MoSe2 as an electroactive substrate can remarkably accelerate the generation of TPrA•+ radicals to react with electrooxidized Ru(bpy)32+, which achieves about 3.4-fold stronger ECL intensity. Thus, an enhanced ECL immunoassay method can be achieved after Ru(bpy)32+-doped silica nanoparticle labeled antibody (Ab2-Ru@SiO2) is captured to the modified electrode via immunological recognition. Using methylated DNA as a target, the immunosensor was prepared by binding capture DNA on AuNPs@MoSe2 modified electrode to successively capture the target, anti-5-methylcytosine antibody (anti-5mC) and Ab2-Ru@SiO2. The proposed strategy could detect 0.26 fM 5 mC (3σ) with a detectable concentration range of 1.0 fM - 10 nM at methylated DNA. This immunosensor showed excellent selectivity, good stability and reproducibility, and acceptable recovery, indicating the broad prospects of the novel coreaction accelerator in clinical diagnosis.

Electrochemiluminescent (ECL) biosensor for Burkholderia pseudomallei based on cobalt-doped MOF decorated with gold nanoparticles and N-(4-aminobutyl)-N-(ethylisoluminol).

A multifunctional catalytic nanomaterial (Co-MOF@AuNP@ABEI) composed of cobalt-doped metal-organic frameworks (Co-MOF), gold nanoparticles (AuNP), and N-(4-aminobutyl)-N-(ethylisoluminol) (ABEI) is reported. Co-MOF@AuNP@ABEI exhibits high synergistic and zero-distance catalytic properties, which are beneficial to the improvement of the detection sensitivity of an electrochemiluminescent (ECL) biosensor. After coupling with the ECL system and 3D magnetic walking nanomachine amplification strategy, the Co-MOF@AuNP@ABEI can achieve an ultrasensitive ECL assay of Burkholderia pseudomallei with the limit of detection (LOD) of 60.3 aM, which is 2 and 4 orders of magnitude lower than individual ECL system without the nanomachine (4.97 fM) and individual walking nanomachine (340 fM), and superior to the pathogenic bacteria analyses in the previous report. Moreover, the LOD of the proposed ECL detection system for the determination of B. pseudomallei in serum sample was as low as 9.0 CFU mL-1. The relative standard deviations (RSD) of ECL intensity for the detection of five B. pseudomallei-spiked serum samples were 4.02%, 0.84%, 0.84%, 1.55%, and 0.21%, respectively. The recoveries of the ECL biosensor for the detection of B. pseudomallei DNA-spiked serum samples were 93.63 ~ 107.83%. Therefore, this work demonstrated that the developed multifunctional catalytic nanomaterial with synergistic and zero-distance catalytic properties can be used as excellent ECL signal reporter to improve the detection sensitivity of ECL biosensor.

A novel lytic phage potentially effective for phage therapy against Burkholderia pseudomallei in the tropics.

BACKGROUND: Burkholderia pseudomallei is a tropical pathogen that causes melioidosis. Its intrinsic drug-resistance is a leading cause of treatment failure, and the few available antibiotics require prolonged use to be effective. This study aimed to assess the clinical potential of B. pseudomallei phages isolated from Hainan, China. METHODS: Burkholderia pseudomallei strain (HNBP001) was used as the isolation host, and phages were recovered from domestic environmental sources, which were submitted to the host range determination, lytic property assays, and stability tests. The best candidate was examined via the transmission electron microscope for classification. With its genome sequenced and analyzed, its protective efficacy against B. pseudomallei infection in A549 cells and Caenorhabditis elegans was evaluated, in which cell viability and survival rates were compared using the one-way ANOVA method and the log-rank test. RESULTS: A phage able to lyse 24/25 clinical isolates was recovered. It was classified in the Podoviridae family and was found to be amenable to propagation. Under the optimal multiplicity of infection (MOI) of 0.1, an eclipse period of around 20 min and a high titer (1012 PFU/ml) produced within 1 h were demonstrated. This phage was found stabile at a wide range of temperatures (24, 37, 40, 50, and 60 °C) and pH values (3-12). After being designated as vB_BpP_HN01, it was fully sequenced, and the 71,398 bp linear genome, containing 93 open reading frames and a tRNA-Asn, displayed a low sequence similarity with known viruses. Additionally, protective effects of applications of vB_BpP_HN01 (MOI = 0.1 and MOI = 1) alone or in combination with antibiotics were found to improve viability of infected cells (70.6 ± 6.8%, 85.8 ± 5.7%, 91.9 ± 1.8%, and 96.8 ± 1.8%, respectively). A significantly reduced mortality (10%) and a decreased pathogen load were demonstrated in infected C. elegans following the addition of this phage. CONCLUSIONS: As the first B. pseudomallei phage was isolated in Hainan, China, phage vB_BpP_HN01 was characterized by promising lytic property, stability, and efficiency of bacterial elimination during the in vitro/vivo experiments. Therefore, we can conclude that it is a potential alternative agent for combating melioidosis.

Highly sensitive fluorescence biosensing of BCR-ABL1 fusion gene based on exponential transcription-triggered hemin catalysis.

Simple, sensitive and specific detection of the transcription level of BCR-ABL1 mRNA possesses vital clinical significance in diagnosis and treatment of chronic myeloid leukemia (CML). In this study, an innovative fluorescence biosensing methodology has been developed for sensitive and specific detection of BCR-ABL1 mRNA by integrating high-efficiency of exponential transcription and superior catalytic performance of DNA-grafted hemin. Exponential transcription was triggered by BCR-ABL1 mRNA to produce plenty of RNA products. They can specifically hybridize with circular dual-labeled hemin (DLH) probe to dissociate the intramolecular hemin dimmers into highly active hemin monomers for catalyzing fluorescence substrate tyramine. This exponential transcription-triggered hemin catalysis (ET-HC) strategy showed highly sensitive and specific for BCR-ABL1 detection with a limit of detection at 0.5 aM and a good linear range from 2 aM to 200 fM. This method was successfully applied to directly detect as low as 0.001% e13a2 transcript isoforms from complex genomic RNA extraction. Compared with clinical routine, the overall process is a thermostatic reaction and eliminates additional reverse transcription operation. Therefore, the developed ET-HC strategy might provide a promising alternative tool for precise diagnosis and personalized treatment of CML.

One-step discrimination of BCR/ABLp210 transcript isoforms directly from RNA extraction with fusion-triggered rolling circle amplification.

BCR/ABLp210 fusion gene, the characteristic biomarker of chronic myelogenous leukemia (CML), contains two different transcription isoforms, e13a2 and e14a2, which lead to differences in the pathological features and response to targeted drug. At present, there is short of simple and fast technology to distinguish these two transcript isoforms. In this paper, RNA fusion-triggered rolling circle amplification (RF-RCA) strategy was developed to distinguish e13a2 and e14a2 transcripts directly from RNA extraction in one step. The simultaneous binding of dumbbell template and corresponding primer with target fused RNA can induce their proximal hybridization and trigger the RCA to produce lots of tandem repeat G-quadruplexes sequences for real time fluorescence readout with the interaction of Thioflavin T and G-quadruplex. The proposed strategy can detect as low as 0.1 aM target and discriminate e13a2 (0.01%) and e14a2 (0.1%) transcript isoforms directly from complex genomic RNA extraction, proving high sensitivity and specificity. Furthermore, the RF-RNA was successfully applied to analyze BCR/ABLp210 isoforms from clinical samples for accurately molecular subtyping and monitoring the response of imatinib treatment. The developed RF-RCA strategy presented an ultrasensitive, accurate and pragmatic toolbox to simple and rapid discriminate BCR/ABLp210 fusion isoforms for promoting clinical research and personalized treatment of CML.

Prediction of the anti-inflammatory mechanisms of curcumin by module-based protein interaction network analysis.

Curcumin, the medically active component from Curcuma longa (Turmeric), is widely used to treat inflammatory diseases. Protein interaction network (PIN) analysis was used to predict its mechanisms of molecular action. Targets of curcumin were obtained based on ChEMBL and STITCH databases. Protein-protein interactions (PPIs) were extracted from the String database. The PIN of curcumin was constructed by Cytoscape and the function modules identified by gene ontology (GO) enrichment analysis based on molecular complex detection (MCODE). A PIN of curcumin with 482 nodes and 1688 interactions was constructed, which has scale-free, small world and modular properties. Based on analysis of these function modules, the mechanism of curcumin is proposed. Two modules were found to be intimately associated with inflammation. With function modules analysis, the anti-inflammatory effects of curcumin were related to SMAD, ERG and mediation by the TLR family. TLR9 may be a potential target of curcumin to treat inflammation.

[Simultaneous determination of beta-elemene, curcumol, germacrone and neocurdione in volatile oil of Curcuma phaeocaulis and vinegar products by GC-MS].

This study aims to develop a method for determination of beta-elemene, curcumol, germacrone and neocurdione in the volatile oil of Curcuma phaeocaulis, and to provide the basis of the quality control method for the volatile oil of C. phaeocaulis and the related preparations. Based on GC-MS, the 4 main compounds were simultaneously determined, with the internal standard n-tridecane. The Agilent 19091S-433 column (0.25 microm x 250 microm x 30 m) was adopted at the temperature of 250 degrees C, the programmed temperature method (60 degrees C for 1 min, 5 degrees C x min x to 110 degrees C for 5 min, 1 degrees C x min(-1) to 140 degrees C, 5 degrees C x min(-1) to 160 degrees C, 10 degrees C x min(-1) to 240 degrees C) was used. Helium gas was used as the carrier gas at a constant flow rat of 1 mL x min(-1), with an injection volume of 1 RL. Mass spectra were taken at 70 eV; the ion-source temperature was 200 degrees C. The relation time and character acteristic ions for each target compound were determined by full scan mode and SIM, and m/z 85.1, 93.1, 121.1, 107.1 and 180.1 were the detection ions of n-tridecane, beta-elemene, curcumol, germacrone and neocurdione. As a result, beta-elemene, curcumol, germacrone and neocurdione were all detected with good separation. They were all in a good linear relationship within each concentration scope. The average recovery rates were in the range of 98.2%-101%. So, the method can be used to control the quality of the volatile of C. phaeocaulis Val. and the preparations related.