Visual Readout Research Articles

Streamside detection of Chinook salmon (Oncorhynchus tshawytscha) environmental DNA with CRISPR technology

AbstractThere is a rapidly growing interest by resource managers to utilize environmental DNA technology (eDNA) as a tool to enhance current management efforts. However, the technology remains relatively specialized, since it requires specific expertise and equipment to perform. To begin to overcome some of the obstacles restricting the widespread, routine adoption of eDNA technology, we evaluated the use of CRISPR Cas12a detection technology for in‐the‐field eDNA detection using Chinook salmon (Oncorhynchus tshawytscha) as a target species. By targeting a highly variable region in the salmonid mitochondrial DNA D‐loop, we were able to demonstrate that CRISPR Cas12a detection technology is both sensitive and specific for Chinook salmon eDNA. Engineering of the technology to work in the field was accomplished by employing rapid eDNA purification and visual readout of results using visual lateral flow or fluorescent detection methods. The technology was piloted on the fall Chinook salmon run in the Snake River of Washington State, USA, and proved to be a viable approach for streamside eDNA monitoring. With the improvement of the technology, CRISPR eDNA detection methods hold great promise in expanding the reach of eDNA as a commonly used resource management tool.

Prognostic role of quantitative [18F]FDG PET/CT parameters in adrenocortical carcinoma

PurposeWe aimed to evaluate the prognostic potential of baseline [18F]FDG PET/CT for overall survival (OS) in patients with adrenocortical carcinoma (ACC).MethodsWe performed a retrospective analysis of 67 treatment-naïve ACC patients with available [18F]FDG PET/CT at time of initial diagnosis. Pretherapeutic PETs of primary tumors were manually segmented and quantitative parameters (maximum/mean/peak standardized uptake value (SUVmax/mean/peak), metabolic tumor volume (MTV) and tumor lesion glycolysis (TLG, defined as TV*SUVmean) were derived. Based on a visual read, absence (M0) or presence of metastatic disease (M1) were evaluated. Kaplan–Meier and Cox regression analyses were used to determine the prognostic value of the above mentioned markers on overall survival adjusted for established prognostic markers.Results24/67 patients (36%) presented with M0 based on PET/CT, while the remaining 43/67 (64%) had M1-status. 32/67 patients died during follow-up and median OS was 48 months. In 12% of patients FDG-PET detected additional metastatic lesion not clearly visible by CT only. In univariable analysis, all quantitatively derived PET parameters failed to reach significance (P ≥ 0.1), and only PET/CT-based M1-status and Ki-67 were associated with increased mortality (M1: HR 13.89, 95% CI 4.15–86.32, P < 0.001; Ki-67 HR 1.29, 95% CI 1.16–1.42; P < 0.0001). Using multivariable Cox regression analyses, M1-status (HR 9.69, 95% CI 2.82–60.99) and Ki-67 index (HR 1.29, 95% CI 1.13–1.04; P < 0.05) remained significant associated with OS.ConclusionIn treatment-naïve ACC patients, the quantitative PET parameter failed to predict OS, but presence of metastases detected by [18F]FDG PET/CT and Ki-67 index were independently associated with shorter OS. Therefore, a simple visual PET-based read-out is of prognostic value at initial diagnosis, while time-consuming PET-based quantification can be omitted.

A field diagnostic method for rapid and sensitive detection of mpox virus.

The mpox outbreak has subdued with fewer reported cases at the present in high-income countries. It is known that mpox virus (MPXV) infection has been epidemic for more than 50 years in African countries. The ancestral MPXV strain has changed into multiple clades, indicating the ongoing evolution of MPXV, which reflects the historical neglect of mpox in Africa, especially after smallpox eradication, and bestows the danger of more severe mpox epidemics in the future. It is thus imperative to continue the development of mpox diagnostics and treatments so we can be prepared in the event of a new mpox epidemic. In this study, we have developed an MPXV detection tool that leverages the recombinase-aid amplification assay by integrating lateral flow strips (RAA-LF) and one-step sample DNA preparation, with visible readout, no need of laboratory instrument, and ready for field deployment. The detection limit reaches 10 copies per reaction. The performance of our RAA-FL assay in diagnosing mpox clinical samples is on par with that of the quantitative polymerase chain reaction (PCR) assay. Taken together, we have developed a point-of-care RAA-LF method of high accuracy and sensitivity, readily deployable for field detection of MPXV. This diagnostic tool is expected to improve and accelerate field- and self-diagnosis, allow timely isolation and treatment, reduce the spread of MPXV, thus effectively mitigate MPXV outbreak in the future.

SARS-CoV-2 recombinase polymerase amplification assay with lateral flow readout and duplexed full process internal control.

Nucleic acid amplification tests for the detection of SARS-CoV-2 have been an important testing mechanism for the COVID-19 pandemic. While these traditional nucleic acid diagnostic methods are highly sensitive and selective, they are not suited to home or clinic-based uses. Comparatively, rapid antigen tests are cost-effective and user friendly but lack in sensitivity and specificity. Here we report on the development of a one-pot, duplexed reverse transcriptase recombinase polymerase amplification SARS-CoV-2 assay with MS2 bacteriophage as a full process control. Detection is carried out with either real-time fluorescence or lateral flow readout with an analytical sensitivity of 50 copies per reaction. Unlike previously published assays, the RNA-based MS2 bacteriophage control reports on successful operation of lysis, reverse transcription, and amplification. This SARS-CoV-2 assay features highly sensitive detection, visual readout through an LFA strip, results in less than 25 minutes, minimal instrumentation, and a useful process internal control to rule out false negative test results.

Recent advances in the CRISPR/Cas system-based visual detection method.

Currently, various infectious pathogens and bacterial toxins as well as heavy metal pollution pose severe threats to global environmental health and the socio-economic infrastructure. Therefore, there is a pressing need for rapid, sensitive, and convenient visual molecular detection methods. The rapidly evolving detection approach based on clustered regularly interspaced short palindromic repeats (CRISPR)/associated nucleases (Cas) has opened a new frontier in the field of molecular diagnostics. This paper reviews the development of visual detection methods in recent years based on different Cas and analyzes their advantages and disadvantages as well as the challenges of future research. Firstly, different CRISPR/Cas effectors and their working principles in the diagnosis of various diseases are briefly reviewed. Subsequently, the article focuses on the development of visual readout signals in point-of-care testing using laboratory-based CRISPR/Cas technology, including colorimetric, fluorescence, and lateral flow analysis. Finally, the challenges and prospects of visual detection methods based on CRISPR/Cas technology are discussed.

Assessment of Reader Technologies for Over-the-Counter Diagnostic Testing.

Background: Over-the-counter (OTC) diagnostic testing is on the rise with many in vitro diagnostic tests being lateral flow assays (LFAs). A growing number of these are adopting reader technologies, which provides an alternative to visual readouts for results interpretation, allowing for improved accessibility of OTC diagnostics. As the reader technology market develops, there are many technologies entering the market, but no clear, single solution has yet been identified. The purpose of this research is to identify and discuss important parameters for the assessment of LFA reader technologies for consideration by manufacturers or researchers. Methods: As part of The National Institute of Biomedical Imaging and Bioengineering's Rapid Acceleration of Diagnostics (RADx) Tech program, reader manufacturers were interviewed to investigate the current state of reader technology development through several parameters identified as important industry standards. Readers were categorized by technology type and parameters including cost, detection method, multiplex capabilities, assay type, maturity, and use case were all assessed. Results: Fifteen reader manufacturers were identified and interviewed, and information on a total of 19 technologies was assessed. Reader technology type was found to be predictive of other attributes, whether the reader is smart technology only, a standalone reader, a reader with smart technology required, or a reader with smart technology optional. Conclusions: Pairing reader technology with OTC diagnostic tests is important for improving existing COVID-19 tests and can be utilized in other diagnostics as the OTC use case grows in popularity. Reader technology type, which is predictive of core reader attributes, should be considered when selecting a reader technology for a specific LFA test within the context of regulatory guidance. As diagnostics increase in complexity, readers provide solutions to accessibility challenges, facilitate public health reporting, and ease the transition to multiplex testing, therefore increasing market availability.

Visually distinguishing between tumor tissue and healthy tissue within ten minutes using proteolytic probes

Fluorescence-emitting probes designed to detect furin, a biomarker protease that is upregulated in tumor tissue, simplifying complex diagnostic workflows and enabling tissue-conserving surgery through direct visual readout.

Urine-based point-of-care testing for factor-Xa-inhibitors in acute ischemic stroke patients: a feasibility study.

Direct oral anticoagulants (DOACs) have become widely used in clinical practice for preventing thromboembolic events. Point-of-care testing methods, particularly those based on urine samples, offer a promising approach for rapid and accurate assessment of DOAC presence. This pilot study aims to evaluate the utility of a urine-based DOAC dipstick test as a point-of-care tool for identifying DOAB presence in acute ischemic stroke (AIS) or transient ischemic attack (TIA) patients. This prospective pilot study included patients with AIS/TIA eligible for DOAC-measurement. After exclusion of 3 patients, 23 patients with DOAC-intake (DOAC group; factor-Xa-inhibitors; n = 23) and 21 patients without DOAC-intake (control-group) remained for analyses. The urine-based DOAC dipstick test and parallel blood-based specific DOAC-level assessment were performed in all patients. Time-intervals of sampling urine/blood sampling and result of DOAC-test were recorded to analyze a potential time benefit based on dipstick evaluation. The urine-based DOAC dipstick test demonstrated high sensitivity (100%) and specificity (100%), correctly identifying all patients with anticoagulatory activity due to DOAC intake (i.e., anti-Xalevel ≥30 ng/mL). Moreover, the visual readout of the test provided semiquantitative information on drug-specific anti-Xa levels, showing a sensitivity of 83% and specificity of 93% to detect anti-Xa levels ≥120 ng/mL. The dipstick test exhibited a median time-benefit of 2:25 h compared to standard blood-based DOAC-level testing. The results of this pilot study underline the efficacy of urine-based point-of-care testing as a rapid and reliable method for assessing DOAC presence in patients with acute ischemic stroke. The value of this tool for clinical decision-making in stroke management needs to be established in future trials.Clinical Trial Registration: Clinicaltrails.org identifier [NCT06037200].

875. Development of a Cheap, Portable Assay to Rapidly Profile Resistant Bacterial Infections

Abstract Background Antimicrobial resistant (AMR) bacterial infections pose a significant and growing health threat worldwide. The burden of these resistant infections is often most severe in lower income areas, where the medical infrastructure necessary for their diagnosis is lacking. The development of cost-effective and accessible diagnostic tools for the rapid identification of resistant infections in low-resource settings is crucial. Specific High-sensitivity Enzymatic Reporter Unlocking, or SHERLOCK, is a promising CRISPR-based system that can aid in the diagnosis of infections and support clinical decision making. Here, we present an assay termed BADLOCK (Bacteria and AMR Detection by SHERLOCK) that uses SHERLOCK technology to rapidly identify bacterial species and AMR genes on blood cultures. SHERLOCK Schematic Enzymatic activity of the Cas protein is turned on when the target gene is bound, which then activates reporter probes. Methods We combine isothermal amplification of gene targets with SHERLOCK-based detection into a single step, allowing for a streamlined workflow and a total time-to-detection of less than two hours. The assay targets the topA gene for species identification, which is highly conserved within species but has sufficient variability between them to allow for discrimination between even closely related pathogens. A panel of clinically important AMR genes, including CTX-M-15, mecA, and five canonical carbapenemases, is also targeted to provide information on resistance profiles. BADLOCK Targets Gene targets included in the BADLOCK assay. Results BADLOCK has high sensitivity and specificity for both AMR genes and species identification and leverages a lateral-flow platform to provide a simple visual readout. We are currently validating BADLOCK on a library of several hundred clinical isolates with known genetic backgrounds, including a large collection of carbapenem-resistant Enterobacterales, which are major contributors to the global burden of resistant infections. Lateral Flow Assay Results Lateral flow results. Strip 1 is the topA gene specific to K. pneumoniae. Strips 2-4 are AMR genes KPC, CTX-M-15, and MecA. Lane 5 is a positive control; lane 6 is a NTC with bottom banding. “+” and “-” symbols mark representative readouts. Conclusion We have developed an assay for the cheap, rapid detection of bacterial infections of the bloodstream capable of detecting common pathogens as well as a panel of AMR genes. Our work has the potential to improve the diagnostic landscape for resistant infections in low-and-middle-income countries, which would represent a major step forward in infectious disease treatment in these underserved areas. Disclosures All Authors: No reported disclosures

Light-Driven Photocatalytic-Photothermal Synergetic System for Portable and Sensitive Nucleic Acid Quantification.

Photosensitizers and photothermal agents have attracted increasing attention for in vitro diagnosis, but the combination remains challenging. Herein, a light-driven photocatalytic-photothermal synergetic system integrated microfluidic distance-based analytical device (PCPT-μDAD) for visual, portable, sensitive, and quantitative detection of targets was developed. Target DNA was recognized and initiated the hybridization chain reaction to form a double-stranded DNA/SYBR Green I (dsDNA/SG-I) complex. By applying the photosensitization of the dsDNA/SG-I complex and the photothermal effect of oxidized 3,3',5,5'-tetramethylbenzidine, the target concentration can effectively translate into a visual distance signal readout. Importantly, the light-driven PCPT-μDAD greatly improves the controllability of catalytic reactions and signal amplification efficiency. The light-driven PCPT-μDAD shows a low limit of detection (fM level), good stability, and high reproducibility for nucleic acid detection.

Simultaneous detection of Pb2+ and Cu2+ based on catalytic etching of AgNTs@AuNHs colorimetric nanoprobe

A colorimetric gold nanohexagonals coated silver nanotriangles (AgNTs@AuNHs) nanoprobe has been constructed for the ultrasensitive detection of trace Pb2+ and Cu2+, based on the fact that Pb2+ and Cu2+ as the catalyzer can selectively enhance the etching rate of AgNTs@AuNHs by S2O32−. In the presence of Pb2+, the inner AgNTs within the AgNTs@AuNHs undergo complete etching to form the hollow AuNHs, leading to the marked decrease in both absorbance and chrominance at 585 nm. In contrast, Cu2+ initiates an etching reaction on the AgNTs, subsequently extending to the AuNHs. These transformations give rise to the formation of hollow gold nanotriangles, accompanied by pronounced red-shifts in the absorption peaks of the detection system. The distinct optical responses induced by Pb2+ and Cu2+ in the AgNTs@AuNHs nanoprobe enable the sensitive and selective detection and quantification of these heavy metal ions. Furthermore, we successfully extracted RGB data from the color changes of AgNTs@AuNHs nanoprobe, enabling a straightforward visual readout for detection of Pb2+ and Cu2+. This method yields satisfactory recoveries when applied to the analysis of Pb2+ and Cu2+ in river water, rice, and onion samples, with detection results closely matching those obtained using ICP-MS. These results collectively underscore the practical applicability of the AgNTs@AuNHs nanoprobe.

B-243 Utilizing Lyophilized LAMP Reagents in Rapid Molecular Assays

Abstract Background Loop Mediated Isothermal Amplification (LAMP) has become a widely used method for detection of target nucleic acids (DNA and RNA) as it offers a robust and simple alternative to PCR. LAMP is particularly well suited to point of care (POC) applications such as COVID-19 diagnostics because only a single incubation temperature is required for nucleic acid amplification and the technology is compatible with simple detection strategies, including colorimetric or lateral flow readouts. To further increase the utility of LAMP in POC molecular diagnostic workflows, elimination of cold chain requirements for reagent shipment and storage is desired. In this study, we investigate single and multiplex detection of several viral infectious diseases using lyophilized RT-LAMP reagents. Methods Assays targeting SARS-CoV-2, FluA, FluB and a host control nucleic acid were evaluated using various detection methods with lyophilized RT-LAMP reagents. Compatibility was established with endpoint LAMP detection strategies, such as colorimetric dyes, and real time multiplex LAMP detection, which is increasingly finding utility in POC testing for common respiratory infections that present with similar symptoms. Results Assays employing lyophilized RT-LAMP reagents maintained robust detection of viral respiratory targets within 30 min at 65°C using various detection strategies. Colorimetric dyes such as eriochrome back T and calcein resulted in successful visual readout upon amplification while, one prominent colorimetric dye, hydroxy naphthol blue, was found to be incompatible with the lyophilized reagents used in the study. Additionally, simple hybridization probes permitted simultaneous detection of SARS-CoV-2, FluA and FluB in a single reaction. Conclusion LAMP is a robust, simple, isothermal amplification strategy that continues to enable testing beyond traditional laboratory settings. Lyophilized reagents extend the utility of LAMP in POC settings regardless of the detection strategy and will be important factors in the extension of this powerful molecular diagnostic tool to decentralized, field, and at-home testing applications.

B-213 Colorimetric LAMP as an Effective Tool for Rapid Molecular Diagnostics

Abstract Background LAMP has become a standard molecular diagnostic technique, and its robustness and flexibility have enabled rapid, simple tests outside of traditional laboratory settings. The need for additional diagnostic methods became urgent during the SARS-CoV-2 pandemic, and LAMP was extensively evaluated and validated for numerous testing applications. We have extended the utility of LAMP to enable colorimetric detection, multiplex assays, variant identification, and robust direct tests for point of-care and workplace surveillance testing. Methods We utilized fluorescent and colorimetric RT-LAMP for assay development and our workplace screening program. A simple lysis buffer was applied to whole saliva samples and added directly to our colorimetric LAMP SARS-CoV-2 tests, with more than 100 000 samples tested. For variant calling we developed novel hybridization probes with single-base sensitivity to regions of sequence changes, and we characterized more than 5000 LAMP assays internally positioning mismatches in the LAMP primer regions to ensure robustness to and tolerance of mutations. Results The colorimetric LAMP assay displayed a sensitivity of 97% and specificity of 100%, and during its utilization in our in-house CLIA-certified workplace screening has identified &amp;gt;300 unique positive cases derived from all variants of concern. 3654 blinded samples were analyzed by 3 operators and colorimetric readout was found to have a strong concordance with real-time fluorescence measurement (kappa = 0.999). Conclusion RT-LAMP is remarkably tolerant of sequence variation, with no individual positional mutation significantly affecting assay performance. Simple visual readout can be utilized for a reliable diagnostic indicator when sophisticated instrumentation is not available or desired.

Violet phosphorene nanosheets coupled with CRISPR/Cas12a in a biosensor with a low background signal for onsite detection of tigecycline-resistant hypervirulent Klebsiella pneumoniae

CRISPR/Cas12a-based detection has been widely used for DNA targets, but always suffers from a relatively high background fluorescent signal, which potentially gives false positive results, and limits the accuracy and specificity of detection. To address this problem, violet phosphorene nanosheets (VPNSs) with excellent DNA adsorption and light absorption capacities were synthesized by liquid exfoliation from bulk violet phosphorus. The introduction of VPNSs into CRISPR/Cas12a-based detection decreased the relatively high background fluorescent signal by 32.7 %, and resulted in a background with the relative fluorescence unit of 2590.5 that was no longer visible via a visual readout, demonstrating that VPNSs are efficient fluorescence quenchers. By combining the detection with a smartphone-based fluorescence imaging system (FIS), a biosensor was developed for simultaneous detection of virulence genes iucA, iroB, peg-344, rmpA and rmpA2, and resistance gene tet(A) carried by tigecycline-resistant hypervirulent Klebsiella pneumoniae (HvKP). Upon optimization, the VPNSs-CRISPR/Cas12a-FIS biosensor detected 1 cfu of tigecycline-resistant HvKP in 60 min with a low background signal. The whole process was performed with portable devices including a block heater and an FIS, and the signal could be observed with either a smartphone or the naked eye. The biosensor provides new insights for exploration of low background, multiple target, and onsite analytical methods based on CRISPR/Cas12a.

Multiplex lateral flow test strip for detection of carbapenemase genes using barcoded tetrahedral DNA capture probe-based biosensing interface.

Carbapenem-resistant Enterobacterales pose significant global health challenges due to their rapid spread and ability to hydrolyse various beta-lactam antibiotics. Rapid tests for these carbapenemase genes are crucial to ensure appropriate prescription administration and infection control. In this study, we developed a rapid visual nanodiagnostic platform for multiplexed detection of carbapenemase genes using a lateral flow strip. The nanodiagnostic strip was designed with separate barcoded DNA tetrahedrons for the blaKPC and blaNDM genes. These tetrahedrons were distributed on a nitrocellulose membrane at two different test lines as capture probes. When tested against a panel of carbapenemase genes, the tetrahedral probes captured single-stranded amplicons of asymmetric PCR via strand hybridisation. The amplicons acted as bridging elements, binding the DNA-modified gold nanoparticles to the test line of the strip, resulting in clear visual readouts specific to the blaKPC and blaNDM genes. By employing barcoded tetrahedrons and asymmetric PCR in conjunction with the lateral flow strip, a single diagnostic test enabled the detection of multiple carbapenemase genes. The test yielded results as low as 0.12 fM for blaKPC and 0.05 fM for blaNDM within 75min. Furthermore, the strip effectively identified specific carbapenemase genes in clinical isolates using real-time PCR, antibody-based lateral flow systems for carbapenemase detection, and carbapenemase phenotype experiments. Thus, the strip develop has a high potential for testing blaKPC and blaNDM genes in practice.

Advances in Point-of-Care Testing of microRNAs Based on Portable Instruments and Visual Detection.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that are approximately 22 nt in length and regulate gene expression post-transcriptionally. miRNAs play a vital role in both physiological and pathological processes and are regarded as promising biomarkers for cancer, cardiovascular diseases, neurodegenerative diseases, and so on. Accurate detection of miRNA expression level in clinical samples is important for miRNA-guided diagnostics. However, the common miRNA detection approaches like RNA sequencing, qRT-PCR, and miRNA microarray are performed in a professional laboratory with complex intermediate steps and are time-consuming and costly, challenging the miRNA-guided diagnostics. Hence, sensitive, highly specific, rapid, and easy-to-use detection of miRNAs is crucial for clinical diagnosis based on miRNAs. With the advantages of being specific, sensitive, efficient, cost-saving, and easy to operate, point-of-care testing (POCT) has been widely used in the detection of miRNAs. For the first time, we mainly focus on summarizing the research progress in POCT of miRNAs based on portable instruments and visual readout methods. As widely available pocket-size portable instruments and visual detection play important roles in POCT, we provide an all-sided discussion of the principles of these methods and their main limitations and challenges, in order to provide a guide for the development of more accurate, specific, and sensitive POCT methods for miRNA detection.

Rapid and visual identification of HIV-1 using reverse transcription loop-mediated isothermal amplification integrated with a gold nanoparticle-based lateral flow assay platform.

Human immunodeficiency virus type one (HIV-1) infection remains a major public health problem worldwide. Early diagnosis of HIV-1 is crucial to treat and control this infection effectively. Here, for the first time, we reported a novel molecular diagnostic assay called reverse transcription loop-mediated isothermal amplification combined with a visual gold nanoparticle-based lateral flow assay (RT-LAMP-AuNPs-LFA), which we devised for rapid, specific, sensitive, and visual identification of HIV-1. The unique LAMP primers were successfully designed based on the pol gene from the major HIV-1 genotypes CRF01_AE, CRF07_BC, CRF08_BC, and subtype B, which are prevalent in China. The optimal HIV-1-RT-LAMP-AuNPs-LFA reaction conditions were determined to be 68°C for 35 min. The detection procedure, including crude genomic RNA isolation (approximately 5 min), RT-LAMP amplification (35 min), and visual result readout (<2 min), can be completed within 45 min. Our assay has a detection limit of 20 copies per test, and we did not observe any cross-reactivity with any other pathogen in our testing. Hence, our preliminary results indicated that the HIV-1-RT-LAMP-AuNPs-LFA assay can potentially serve as a useful point-of-care diagnostic tool for HIV-1 detection in a clinical setting.

Efficacy of LAMP assay for Mycobacterial spp. detection to prevent treatment delays and onset of drug resistance: a systematic review and meta-analysis.

Tuberculosis (TB) remains a deadly disease affecting one-third population globally. Long turnaround time and poor sensitivity of the conventional diagnostics are the major impediments for faster diagnosis of Mycobacterial spp to prevent drug resistance. To overcome these issues, molecular diagnostics have been developed. They offer enhanced sensitivity but require sophisticated infrastructure, skilled manpower and remain expensive. In that context, loop-mediated isothermal amplification (LAMP) assay, recommended by the WHO in 2016 for TB diagnosis, sounds as a promising alternative that facilitates visual read outs. Therefore, the aim of the present study is to conduct a meta-analysis to assess the diagnostic efficiency of LAMP for the detection of a panel of Mycobacterium spp. following PRISMA guidelines using scientific databases. From 1600 studies reported on the diagnosis of Mycobacterium spp., a selection of 30 articles were identified as eligible to meet the criteria of LAMP based diagnosis. It was found that most of the studies were conducted in high disease burden nations such as India, Thailand, and Japan with sputum as the most common specimen to be used for LAMP assay. Furthermore, IS6110 gene and fluorescence-based detections ranked as the most used target and method respectively. The accuracy and precision rates mostly varied between 79.2% to 99.3% and 73.9% to 100%, respectively. Lastly, a quality assessment based on QUADAS-2 of bias and applicability was conducted. LAMP technology could be considered as a feasible alternative to current diagnostics considering high burden for rapid testing in low resource regions.

A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application.

Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is the second leading cause of death after COVID-19 pandemic. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform for tuberculosis diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified the specific sdaA gene of MTB by MCDA, and the MCDA results were then decoded by CRISPR-Cas12a-based detection, resulting in simple visual fluorescent signal readouts. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the sdaA gene of MTB. The optimal temperature for MCDA pre-amplification is 67°C. The whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), and CRISPR-Cas12a-gRNA biosensing process (5 minutes). The limit of detection (LoD) of the MTB-MCDA-CRISPR assay is 40 fg per reaction. The MTB-MCDA-CRISPR assay does not cross reaction with non-tuberculosis mycobacterium (NTM) strains and other species, validating its specificity. The clinical performance of MTB-MCDA-CRISPR assay was higher than that of the sputum smear microscopy test and comparable to that of Xpert method. In summary, the MTB-MCDA-CRISPR assay is a promising and effective tool for tuberculosis infection diagnosis, surveillance and prevention, especially for point-of-care (POC) test and field deployment in source-limited regions.

Facile and portable multimodal sensing platform driven by photothermal-controlled release system for biomarker detection

Portable, maneuverable and reliable versatile-integrated analytical devices are urgently demanded but still extremely challenging to meet the requirements of point-of-care testing in resource-limited areas. Herein, a multifunctional sensing platform with excellent photothermal performance was implanted into the miniature zone of a paper-based electrochemiluminescent (ECL) biosensor for accurate detection of interleukin-6, which could flexibly interconnect the visualized distance and temperature readout with ultrasensitive ECL response. Concretely, the multipurpose MBene and TaSe2 composites (MBene@TaSe2) prepared via self-assembly approach as target-associated photothermal element was introduced in the paper-based analytical device (PAD) and served as multi-signals trigger. Under the laser irradiation, MBene@TaSe2 probe not only generated heat for rapid temperature output, but also triggered the phase transition behavior of thermoresponsive poly (N-isopropylacrylamide) (pNIPAM) hydrogel to release loaded malachite green (MG) dye for distance-based visual readout. Simultaneously, the released MG was also utilized as effective quencher to decrease the ECL signal of luminol. Benefitting from this dexterous architecture, the speedy preliminary screening and precise quantitative analysis could be subsequently obtained in single-drop sample through one-step sandwich immunoreaction, which avoids additional separation operations and greatly simplifies the analysis procedure. Undeniably, this work provides ingenious insights for advancing the development of convenient and fast multifunction-integrated PAD in family surveillance and intelligent diagnosis.