Amplification Detection Research Articles

MX/MWCNTs composite material aids new strategy for HBV-DNA electrochemical biosensor: achieving multi-level signal amplification and unlabeled detection

Hepatitis B virus DNA (HBV-DNA) serves as a crucial biomarker for the detection of the hepatitis B virus, with variations in its concentration in blood samples providing vital insights into patient infection and recovery status. We have developed an electrochemical biosensor tailored for HBV-DNA detection, exhibiting high sensitivity, specificity, and a broad detection range. The biosensor utilizes a composite material composed of MXene and multiwalled carbon nanotubes (MX/MWCNTs), prepared through a hand-shaking self-assembly technique, to modify the glassy carbon electrode (GCE) and serve as signal receivers. A meticulously designed padlock probe DNA specifically recognizes HBV-DNA and forms a double-stranded DNA structure, which is subsequently degraded by exonuclease III. The resulting sequence then hybridizes with a PolyT-primer to create template, triggering rolling circle amplification and releasing a substantial amount of pyrophosphate for the initial level of signal amplification. Subsequently, the pyrophosphate induces the release of methylene blue (MB) from MB@ZIF-90 composite, achieving a second level of signal amplification. The released MB is then adsorbed by the MX/MWNCNTs-modified GCE, which possesses cationic dye adsorption capabilities and a large specific surface area, generating a detection signal and completing the third level of amplification. This biosensor offers a detection range spanning from 1.0 pM to 1.0 × 106 pM, with a remarkable detection limit as low as 0.5 pM (3σ/S), and is capable of distinguishing base mismatches. Furthermore, it has demonstrated excellent specificity and recovery performance in serum testing. The performance of this biosensor demonstrates significant potential for clinical application, with the ultimate goal of improving the diagnosis and management of hepatitis B.Graphical

A Chromatography Test Strip of Exonuclease III-Amplified Aptamer for Rapid Identification of Prorocentrum minimum.

Recently, the scale and frequency of harmful algae blooms (HABs) have gradually increased, posing a serious threat to human health, marine ecosystems and economic development. For early warning, a method is required that can quickly detect and monitor microalgae. It is proposed to use aptamer targeted to Prorocentrum minimum, along with exonuclease III (Exo III), gold nanoparticles, target single-stranded DNA and hairpin structure probe to construct a new method, i.e. aptamer-lateral flow dipstick (LFD) based on Exo III-assisted signal amplification assay (ALBEA). The key conditions, including signal amplification and LFD detection, are optimized. Under the optimal conditions, the detection limit of ALBEA was 1.25 cells mL-1. The cross-reactivity test showed no positive result except for P. minimum, indicating that the method is highly specific. The anti-interference test confirmed that the technique was not affected by the presence of other microalgae. The tested results of P. minimum cultured under different nutrient conditions and different growth stages demonstrated that the method is not affected by the cell state. Furthermore, the test results of simulated natural water samples further validated the practicality of the ALBEA. In conclusion, the established ALBEA offers a sensitive, specific and user-friendly tool, which can be used for the rapid detection of P. minimum and also provides a reference for the detection of other microalgae.

A simple and integrated fiber-optic real-time qPCR platform for remote and distributed detection of epidemic virus infection.

Quantitative polymerase chain reaction (qPCR) is a well-recognized technique for amplifying and quantifying nuclear acid, and its real-time monitoring capability, ultrahigh sensitivity, and accuracy make it a "golden-standard" tool in both molecular biology research and clinical diagnostics. However, current qPCR tests rely on bulky instrumentation and skilled laboratorians in centralized laboratories, which spatially and temporally separate the sample collection and test, leading to longer sample turnaround times (TATs) and limited working conditions. Herein, we propose an integrated optical fiber real-time polymerase chain reaction (iF-PCR) system that successfully allows convenient sample collection, rapid thermocycling, closed-loop thermal annealing, and real-time fluorescence detection in a tiny capillary reactor. By leveraging the easy-handling capillary-fiber structure and rapid photothermal actuation of the graphene-decorated fiber head, the whole TAT can be shortened, including sampling and 40 thermocycle amplifications, within 23min, in which an ultrasmall sample volume of ∼15μL is needed. Furthermore, the thermal amplification and fluorescence detection capability of the optical fiber system was cross-checked by a commercial qPCR instrument. The fiber-optic qPCR strategy can correctly distinguish between positive and negative samples of clinical respiratory syncytial virus (RSV) without the need for laboratory professional skills. Taking advantage of the distance signal transmission nature of optical fibers, the proposed strategy enables remote testing, which can eliminate the necessity of instrument deployment in crowded biosafety laboratories and facilitate potential distributed pathogen testing for coping with a new round of pandemics.

High-impedance microwave resonators with two-photon nonlinear effects

Nonlinear effects play a central role in photonics as they form the foundation for most of the device functionalities such as amplification and quantum state preparation and detection. Typically the nonlinear effects are weak and emerge only at high photon numbers with strong drive. Here we present an experimental study of a Josephson junction -based high-impedance resonator. We show that by taking the resonator to the limit of consisting effectively only of one junction, results in strong nonlinear effects already for the second photon while maintaining a high impedance of the resonance mode. Our experiment yields thus resonators with strong interactions both between individual resonator photons and from the resonator photons to other electric quantum systems. We also present an energy diagram technique which enables to measure, identify and analyse different multi-photon optics processes along their energy conservation lines.

Development of a rapid isothermal amplification method for clinical detection of Pseudomonas aeruginosa

ABSTRACT Background Pseudomonas aeruginosa (P. aeruginosa) is widely distributed in air, soil, and water, human respiratory tract, intestinal tract, and skin. It can induce bloodstream infection, urinary tract infection, gastrointestinal tract infection, respiratory tract infection, etc. Conventional bacterial isolation, culture, and identification are time-consuming, and many false negative results, which cannot meet the needs of precise clinical diagnosis，and proper treatment. This study aims to develop a rapid isothermal amplification assay of Pseudomonas aeruginosa. Methods Specific primers were designed according to the National Center for Biotechnology Information (NCBI) database based on the highly conserved sequence of Pseudomonas aeruginosa virulence factor gene lecA, and a recombinase polymerase amplification (RPA) detection method was established. The sensitivity and specificity were calculated, as well as the collection and processing of clinical samples. Results The thermostatic amplification technique for Pseudomonas aeruginosa established in this paper allows nucleic acid detection within 10 minutes without cross-amplification with other bacterial strains. 27 P. aeruginosa infections were accurately detected in 300 clinical samples. Conclusion The rapid detection system based on thermostatic amplification had shown high sensitivity and specificity in this study, indicated that this method can effectively assist clinical bacterial detection.

Multiplex Digital Nucleic Acid Analysis by a LAMP-Argonaute Coupling Assay via a Parallel Droplet Fusion SlipChip.

Multiplex digital nucleic acid analysis (NAA) allows the precise quantification of multiple target nucleic acids with single-molecule sensitivity, making it highly appealing for life science research and clinical diagnostics. Nucleic acid-guided endonucleases, such as CRISPR, have demonstrated great potential in digital NAA. However, performing multiplex digital NAA with an endonuclease remains challenging. The thermophilic Argonaute protein (Ago) enables specific targeting of multiple sequences by a single enzyme, exhibiting superior potential in multiplex detection. Here, we developed a multiplex digital NAA by coupling nucleic acid amplification and Ago-specific detection using parallel droplet fusion facilitated by a SlipChip. The SlipChip can generate a series of droplets to perform multiplex digital loop-mediated isothermal amplification (LAMP), followed by a series of droplets containing Ago reagents for parallel mixing and reactions, resulting in three distinct digital fluorescence signals (FAM, ROX, and Cy5) corresponding to each specific target sequence. We performed viral load analysis of respiratory viruses, including influenza A, influenza B, and SARS-CoV-2, within 60 min. In addition, we used this digital LAMP-Ago assay to analyze viral loads in 34 clinical samples. The system provides a multiplex digital NAA capable of precise nucleic acid quantification with high sensitivity and specificity.

Comparison of Plant Genomic DNA Extraction Kits Using the Proofman-LMTIA Amplification Assay.

The extraction of DNA is the basis of molecular biology research. The quality of the extracted DNA is one of the key factors for the success of molecular biology experiments. To select a suitable DNA extraction method for Chinese medicinal herbs and seeds. In this experiment, four commercial DNA extraction kits were used to extract the genomic DNA (gDNA) from the Pinellia ternata (Thunb.) Breit. powder; Arisaema amurense Maxim. powder as well as the seeds of Glycine max (L.) Merr. On the one hand, the concentration and purity of DNA extracted by these four kits were compared. On the other hand, nucleic acid amplification experiments were performed on three samples extracted by each of the four kits by Proofman-LMTIA methods, which is a novel nucleic acid isothermal amplification technique. The concentration and purity of DNA extracted by different kits were used to determine which methods were suitable for the dry powder of Chinese herbal medicines and seeds. The efficiency of the amplification curve to show whether the extracted DNA can be used in nucleic acid amplification experiments. The results showed that the Proofman-LMTIA methods were of high specificity and the optimal reaction temperatures were 63, 59, and 59°C for P. ternata (Thunb.) Makino; A. amurense Maxim. and G. max (L.) Merr., respectively. The concentration and purity of the gDNAs extracted with all kits were within the acceptable ranges; meanwhile, the amplification of the gDNA extracted by Kit II was of the highest efficiency. In this experiment, the principle, concentration, purity, and time taken for extracting DNA with four kits were compared. The automated extraction kit based on the magnetic method is suitable for extracting DNA from Chinese medicinal herbs and seeds. The extracted DNA is suitable for nucleic acid amplification detection.

Detection of Domain Name Server Amplification Distributed Reflection Denial of Service Attacks Using Convolutional Neural Network-Based Image Deep Learning

Domain Name Server (DNS) amplification Distributed Reflection Denial of Service (DRDoS) attacks are a Distributed Denial of Service (DDoS) attack technique in which multiple IT systems forge the original IP of the target system, send a request to the DNS server, and then send a large number of response packets to the target system. In this attack, it is difficult to identify the attacker because of its ability to deceive the source, and unlike TCP-based DDoS attacks, it usually uses the UDP protocol, which has a fast communication speed and amplifies network traffic by simple manipulating options, making it one of the most widely used DDoS techniques. In this study, we propose a simple convolutional neural network (CNN) model that is designed to detect DNS amplification DRDoS attack traffic and has hyperparameters adjusted through experiments. As a result of evaluating the accuracy of the proposed CNN model for detecting DNS amplification DRDoS attacks, the average accuracy of the experiment was 0.9995, which was significantly better than several machine learning (ML) models in terms of performance. It also showed good performance compared to other deep learning (DL) models, and, in particular, it was confirmed that this simple CNN had the fastest time in terms of execution compared to other deep learning models by experimentation.

Point-of-Care Diagnosis of Tuberculosis Using a Portable Nucleic Acid Test with Distance-Based Readout.

Rapid and accurate diagnosis of tuberculosis (TB) infection in resource-limited settings is critically needed to stop the spread of the disease but remains difficult to achieve. Herein, we report a fast, inexpensive nucleic acid test with distance-based readout (FINDR) for TB. Based on the unique chromatographic behavior of DNA intercalating dye on unmodified cellulose paper, our FINDR platform converted the amplicons of loop-mediated isothermal amplification (LAMP) into the migration distance on a paper-based analytical device. A suite of innovations were further introduced to enhance the accessibility of FINDR, including (1) screening optimal LAMP primers for distance-based readout; (2) developing a chip-on-cover design capable of streamlining LAMP amplification and distance-based detection in a fully sealed FINDR chip with a simple hand-based swirling for liquid transfer; and (3) integrating FINDR with an upstream portable pipetting-free DNA extractor and a downstream smartphone app to simplify sample processing and data interpretation. With these innovations, FINDR demonstrated a high clinical sensitivity (96.6%) and specificity (100%) upon validation against clinical sputum samples. Successful clinical validation was also achieved when FINDR was deployed as a point-of-care test for detecting TB under resource-limited conditions.

An MSRE-Assisted Glycerol-Enhanced RPA-CRISPR/Cas12a Method for Methylation Detection.

Nasopharyngeal carcinoma (NPC) is a malignant tumor with high prevalence in southern China. Aberrant DNA methylation, as a hallmark of cancer, is extensively present in NPC, the detection of which facilitates early diagnosis and prognostic improvement of NPC. Conventional methylation detection methods relying on bisulfite conversion have limitations such as time-consuming, complex processes and sample degradation; thus, a more rapid and efficient method is needed. We propose a novel DNA methylation assay based on methylation-sensitive restriction endonuclease (MSRE) HhaI digestion and Glycerol-enhanced recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection (HGRC). MSRE has a fast digestion rate, and HhaI specifically cleaves unmethylated DNA at a specific locus, leaving the methylated target intact to trigger the downstream RPA-Cas12a detection step, generating a fluorescence signal. Moreover, the detection step was supplemented with glycerol for the separation of Cas12a-containing components and RPA- and template-containing components, which avoids over-consumption of the template and, thus, enhances the amplification efficiency and detection sensitivity. The HGRC method exhibits excellent performance in the detection of a CNE2-specific methylation locus with a (limit of detection) LOD of 100 aM and a linear range of 100 aM to 100 fM. It also responds well to different methylation levels and is capable of distinguishing methylation levels as low as 0.1%. Moreover, this method can distinguish NPC cells from normal cells by detecting methylation in cellular genomes. This method provides a rapid and sensitive approach for NPC detection and also holds good application prospects for other cancers and diseases featuring DNA methylation as a biomarker.

Simultaneous detection of moxifloxacin and gatifloxacin by Cu-TCPP/rGO electrochemical sensor

Moxifloxacin (MOX) and gatifloxacin (GAT) are structurally similar and possess close oxidation peak potentials, making it difficult for simultaneous detection by electrochemical methods. To address this, an electrochemical sensor was constructed with reduced graphene oxide (rGO) and copper porphyrin complex (Cu-TCPP), achieving signal amplification and simultaneous detection of MOX and GAT. The linear relation range from 5.00 to 600.00 μmol·L−1 for MOX (LOD is 1.34 μmol·L−1) and 10 to 300 μmol·L−1 for GAT (LOD is 0.59 μmol·L−1). When applied to water samples, it achieved recoveries of 100.09–112.02 % with a relative standard deviation (RSD) of less than 6 % for MOX, and 97.27–110.72 % with an RSD of less than 4 % for GAT. Further calculations indicated that the electro-oxidation of GAT involved two protons and two electrons.

Validation of a Novel Strategy for Fluorescence Quenching for a Self-Quenching Fluorogenic Probe and Its Application for Visual Loop-Mediated Isothermal Amplification Detection During Food Safety Analysis.

The self-quenching fluorogenic probe facilitates precise identification of LAMP (loop-mediated isothermal amplification) amplicons, unaffected by non-specific products resulting from primer dimers. However, low quenching efficiency by surrounding nucleobases leads to high background signal, posing significant challenges for visual inspection with the naked eye. The present study aims to identify an oligonucleotide sequence that is complementary to the self-quenching fluorogenic probe, and to employ the fluorescence super-quenching mechanism of double-stranded DNA to establish a visualization system for the LAMP assay. The results indicated that the incorporation of a sequence fully complementary to the probe could significantly reduce the system's background fluorescence (p < 0.05). When the melting temperature exceeds room temperature, truncating the complementary sequence from the 3' end does not compromise the probe's quenching efficiency. The LAMP visualization system, using a 10-13-base complementary sequence of the loop primer-based probe, could effectively minimize background fluorescence and yield straightforward visual results post-reaction. Applied to rainbow trout and Atlantic salmon detection, the system detected 1 pg DNA in a closed-tube format. In conclusion, a suitable complementary sequence can reduce the background fluorescence of the self-quenching fluorogenic probe. Employing this sequence alongside the self-quenching fluorogenic probe to develop a low-background fluorescence LAMP system demonstrates great potential for successful visual detection and holds considerable promotional merit.

Analytical Validation and Performance Evaluation of Amplicon-Based Next-Generation Sequencing Assays for Detecting ERBB2 and Other Gene Amplifications in Solid Tumors.

Targeted next-generation sequencing (NGS) panels are increasingly being utilized to identify actionable gene amplifications (copy number > 4) among solid tumors. This study validated the analytical performance of two amplicon-based NGS assays, the Oncomine Comprehensive Panel (OCAv3) and the Oncomine Focus Assay (OFA), for detecting gene amplification in formalin-fixed paraffin-embedded (FFPE) tumors of varying cellularity. OCAv3 was assessed for amplification detection in 756 FFPE samples comprising various tumor types. We demonstrated that with standardized quality control metrics, including median absolute pairwise difference score, these assays can achieve a near-perfect positive predictive value, although their sensitivity for detecting amplifications significantly decreased in tumors with cellularity below 30%. Stratifying tumor cellularity into 10-30%, 31-60%, and 61-95% groups revealed significantly higher gene amplification detection rates in the 31-60% and 61-95% groups versus the 10-30% group (20.6% and 26.7% vs. 9.2%, p < 0.0001). When considering all detected gene amplifications, the average amplification calling per sample was nearly five-fold lower in the 10-30% group versus the 61-95% group (0.11 vs. 0.52; p < 0.0001). To further investigate the analytic performance of OCAv3 in detecting ERBB2 amplification, we analyzed a cohort of 121 uterine carcinomas with confirmed ERBB2 status by HER2 IHC or FISH, in which a threshold incorporating amplifications and tumor cellularity achieved 79% sensitivity and 100% specificity, potentially eliminating the need for FISH analysis in 34% of equivocal cases. In a separate validation cohort, similar analytical performance was observed, with the threshold demonstrating consistent sensitivity and specificity. This study highlights the strengths and limitations of amplicon-based NGS assays in detecting amplifications using real-world data.

Increased mTOR activity and RICTOR copy number in small cell lung carcinoma progression

Small cell lung carcinoma (SCLC) is a highly malignant cancer with early metastatic dissemination and poor clinical outcomes. Mutations in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway, including the frequently occurring rapamycin-insensitive protein (RICTOR) amplification, have been described in these tumours. Moreover, the associated mTOR hyperactivity could be exploited for personalised treatment. Our aim was to study mTOR activity, RICTOR amplification, and their role during SCLC progression. In situ mTOR activity and Rictor expression were characterised by immunohistochemistry in 50 primary and 50 brain metastatic tumours, and 14 paired SCLC patient samples. RICTOR copy number changes were analysed by fluorescence in situ hybridisation of the paired SCLC patient samples and in vivo experiments. Additionally, in vitro sensitivity to cisplatin and mTOR inhibitors was evaluated in SCLC cell lines harbouring RICTOR amplification and other mTOR pathway mutations. High Rictor expression and mTOR complex 2 (mTORC2) hyperactivity were significantly associated with brain metastases and worse overall survival. An increase in RICTOR copy number was observed in paired samples during progression. The importance of these alterations was confirmed both by the sensitising effect of vistusertib in vitro and the RICTOR copy number/expression changes in xenografts. Our study highlights the role of mTORC2 in SCLC progression. Early detection of RICTOR amplification in primary tumours and targeting mTORC2 in these cases may represent a promising novel strategy to develop personalised therapy for metastasis prevention.

Automatic and integrated detection of nucleic acid by using a dual-mode thermal controlled digital microfluidic chip

The detection of nucleic acids, which typically consists of nucleic acid extraction, reverse transcription and amplification, is a crucial component of molecular diagnostics. However, traditional methods have several limitations, including high reliance on skilled personnel, low degree of automation and lengthy assay times. Additionally, these methods also require separate areas for the different steps to avoid contamination. Though a number of digital microfluidic (DMF) chips have been reported to detect the nucleic acids, but there is a noticeable absence of fully integrated DMF platforms capable of concurrently performing nucleic acid extraction, amplification, and signal detection. Here, a DMF chip combined with a dual-mode thermal control module was developed to realize the automatic and integrated detection of nucleic acids. The dual-mode thermal control module provided the required temperatures (with a range from 4 °C to 95 °C) for all the steps of nucleic acid detection. Pseudoviruses of SARS-CoV-2 and monkeypox were chosen to demonstrate the feasibility of the DMF chip, and all assays were completed within 40 min. The results confirmed that the products extracted under 4 °C exhibited high concentration and integrity, which were comparable to those by traditional methods. And the analytical performance of amplify was comparable to that of traditional methods with a more compact design, shorter time, smaller size and more automatic operation. With the ability of dual-mode thermo control and full automatic operation, nucleic acid detection with the characteristics of sample-in-result-out was realized on the DMF chip, which holds great promise in fields such as in vitro diagnostics and point‐of‐care testing.

Advanced, Real-Time Programmable FPGA-Based Digital Filtering Unit for IR Detection Modules

This paper presents a programmable digital filtering unit dedicated to operating with signals from infrared (IR) detection modules. The designed device is quite useful for increasing the signal-to-noise ratio due to the reduction in noise and interference from detector–amplifier circuits or external radiation sources. Moreover, the developed device is flexible due to the possibility of programming the desired filter types and their responses. In the circuit, an advanced field-programmable gate array FPGA chip was used to ensure an adequate number of resources that are necessary to implement an effective filtration process. The proposed circuity was assisted by a 32-bit microcontroller to perform controlling functions and could operate at frequency sampling of up to 40 MSa/s with 16-bit resolution. In addition, in our application, the sampling frequency decimation enabled obtaining relatively narrow passband characteristics also in the low frequency range. The filtered signal was available in real time at the digital-to-analog converter output. In the paper, we showed results of simulations and real measurements of filters implementation in the FPGA device. Moreover, we also presented a practical application of the proposed circuit in cooperation with an InAsSb mid-IR detector module, where its self-noise was effectively reduced. The presented device can be regarded as an attractive alternative to the lock-in technique, artificial intelligence algorithms, or wavelet transform in applications where their use is impossible or problematic. Comparing the presented device with the previous proposal, a higher signal-to-noise ratio improvement and wider bandwidth of operation were obtained.

BIOM-36. DETECTION OF CIRCULATING TUMOR DNA (CTDNA) IN CEREBROSPINAL FLUID (CSF) IN PATIENTS WITH GLIOBLASTOMA TREATED IN PHASE I CLINICAL TRIAL

Abstract BACKGROUND As circulating tumor DNA (ctDNA) has been shown to be a source of tumor-specific biomarkers, liquid biopsy has emerged as a novel noninvasive tool in diagnosis, disease monitoring and treatment selection in several solid tumors. However, its use in brain tumors remains challenging because ctDNA is commonly not detected in blood. Thus, CSF (cerebrospinal fluid) has emerged as a potential source of ctDNA in the context of brain tumors. PATIENTS AND METHODS We prospectively evaluated the detection of ctDNA in CSF (CSF-ctDNA) +/- blood of patients with pre-treated MTAP-deleted glioblastoma, currently treated in a phase I clinical trial evaluating PRMT5 inhibitors. We did CSF samples at C2D15 and at progression, after patient’s oral and written consent. CSF was collected by lumbar puncture and analyzed with a panel of 35 genes including IDH1-2, TERT promoter and EGFR. Blood was collected, synchronously with CSF, by peripheral veinous punction and analyzed with the Foundation One Liquid CDx panel. RESULTS Between January and April 2024, five patients were included. All of them undergone initial surgical resection and received at least one previous systemic treatment associated with radiotherapy. At least one somatic mutation was identified in each patient, including EGFR amplification in 2/5 patients (40%). CSF was not available at baseline. One patient provided CSF only at progression. Four patients provided CSF at C2D15 on treatment, and among them, one patient was also collected at progression. At data cut-off, one patient was still on treatment. We identified a detectable level of CSF-ctDNA in only one patient with occipital tumor localization, with detection of EGFR amplification known at diagnosis. Synchronous blood analysis showed only clonal hematopoiesis variants. CONCLUSION We showed in this small cohort of patients that detection of CSF-ctDNA is technically feasible. The type of panel used might be optimized for this population especially to monitor patients as a potential surrogate biomarker for treatment response.

Conception and Optimization of Extraction-Free Loop-Mediated Isothermal Amplification Detection of Dry Rot Fungus Serpula lacrymans.

The use of nucleic acid-based detection tools for microorganisms and fungi has become a gold standard. This is particularly the case for wood-decaying fungi like Serpula lacrymans, which are hard to discriminate based on macroscopic and microscopic observations. This dry rot is important to detect as it is particularly destructive in an infested building, which requires immediate action to prevent spreading and significant damage to structural elements. Through the development and optimization of loop-mediated isothermal amplification against S. lacrymans-specific rDNA internal transcribed spacer region, we demonstrate that it is possible to achieve rapid and specific amplification without nonspecific self-amplification in a similar range as real-time quantitative PCR without any necessary DNA isolation using a colorimetric detection assay. Through a combined set of self-amplification minimization along with hand-held sample homogenization, the LAMP assay was optimized to provide a femtogram-range assay capable of confirming identification in a real field sample either predominantly composed of S. lacrymans or containing the fungus while remaining negative when tested on different types of fungi found in basement-collected samples.

Transcriptome amplification and fusion gene detection using ultra-trace cells derived from cytology specimens

Tumours are complex and despite scientific breakthroughs, there is much to be elucidated about cancer and its mechanisms. By personalising treatment, outcomes can be improved and genetic analysis is key to this as it allows genetic alterations to be identified and treatment to be precisely targeted. Dr Kenji Amemiya is a cytotechnologist and Chief Clinical Laboratory Technician at the Yamanashi Prefectural Central Hospital Genome Analysis Center (GAC). He is working to better understand the complex inner landscape of cancer and therefore improve cancer detection, diagnosis and treatment. Formalin‐fixed paraffin‐embedded (FFPE) specimens are typically used for genetic testing but there are drawbacks to this, including the FFPE DNA becoming damaged during the fixation process with formalin, resulting in low quality and quantity of DNA. Amemiya is collaborating closely with Chairman of the GAC, Professor Emeritus Masao Omata. They are interested in the use of cytology samples instead of tissue samples in genome analysis, as a less invasive method. Survival rates are increased when genomic analysis is utilised in non-small cell lung cancer (NSCLC). In one project, Amemiya, Omata and the team explored the potential of performing NGS analysis with archived cytological specimens (ACSs). They utilised an integrated NGS platform called the Oncomine Dx Target Test Multi-CDx system to compare the quantity and quality of extracted nucleic acids, the sequencing quality control (QC) and the detected variants between ACSs and FFPE tissues. They found that the RNA integrity was higher in ACSs and there was no difference in sequencing results, compared with FFPE tissues.

A novel machine-learning aided platform for rapid detection of urine ESBLs and carbapenemases: URECA-LAMP.

Pathogenic gram-negative bacteria frequently carry genes encoding extended-spectrum beta-lactamases (ESBL) and/or carbapenemases. Of great concern are carbapenem resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. Despite the need for rapid AMR diagnostics globally, current molecular detection methods often require expensive equipment and trained personnel. Here, we present a novel machine-learning-aided platform for the rapid detection of ESBLs and carbapenemases using Loop-mediated isothermal Amplification (LAMP). The platform consists of (i) an affordable device for sample lysis, LAMP amplification, and visual fluorometric detection; (ii) a LAMP screening panel to detect the most common ESBL and carbapenemase genes; and (iii) a smartphone application for automated interpretation of results. Validation studies on clinical isolates and urine samples demonstrated percent positive and negative agreements above 95% for all targets. Accuracy, precision, and recall values of the machine learning model deployed in the smartphone application were all above 92%. Providing a simplified workflow, minimal operation training, and results in less than an hour, this study demonstrated the platform's feasibility for near-patient testing in resource-limited settings.IMPORTANCEExtended-spectrum beta-lactamases (ESBL) and carbapenemases confer resistance to third-generation cephalosporins and carbapenems in pathogenic Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. Conventional antimicrobial susceptibility testing is based on phenotypic methods, and results can take several days to be obtained. Current genotypic detection methods can be rapid but require expensive equipment and trained personnel. In this study, we present a novel machine learning-aided platform for the rapid detection of ESBLs and carbapenemases using Loop-mediated isothermal Amplification (LAMP). The validation of the platform demonstrated percent positive and negative agreements above 95% for all targets. The newly developed platform provided a simplified workflow, minimal technical training, and results in less than an hour. This study demonstrated the platform's feasibility for rapid testing of ESBL and carbapenemases in bacteria and urine specimens.