Rapid Nucleic Acid Research Articles

Evaluation of a Point-of-Need Molecular Diagnostic Tool Coupled with Rapid DNA Extraction Methods for Visceral Leishmaniasis.

A rapid, cost-effective, and simple nucleic acid isolation technique coupled with a point-of-need DNA amplification assay is a desirable goal for programmatic use. For diagnosis of Visceral Leishmaniasis (VL), Recombinase Polymerase Amplification (RPA) rapid tests for the detection of Leishmania DNA are versatile and have operational advantages over qPCR. To facilitate the delivery of the RPA test at point-of-need for VL diagnosis, we compared two rapid DNA extraction methods, SwiftDx (SX) and an in-house Boil and Spin (BS) method, coupled with RPA amplification, versus more widely used methods for DNA extraction and amplification, namely Qiagen (Q) kits and qPCR, respectively. A total of 50 confirmed VL patients and 50 controls, matched for age and gender, were recruited from Mymensingh, Bangladesh, a region highly endemic for VL. Blood samples were collected from each participant and DNA was extracted using Q, SX and BS methods. Following DNA extraction, qPCR and RPA assays were performed to detect L. donovani in downstream analysis. No significant differences in sensitivity of the RPA assay were observed between DNA extraction methods, 94.00% (95% CI: 83.45-98.75%), 90% (95% CI: 78.19-96.67%), and 88% (95% CI: 75.69-95.47%) when using Q, SX, and BS, respectively. Similarly, using qPCR, no significant differences in sensitivity were obtained when using Q or SX for DNA extraction, 94.00% (95% CI: 83.45-98.75%) and 92.00% (80.77-97.78%), respectively. It is encouraging that RPA and qPCR showed excellent agreement (k: 0.919-0.980) when different extraction methods were used and that the DNA impurities using BS had no inhibitory effect on the RPA assay. Furthermore, significantly higher DNA yields were obtained using SX and BS versus Q; however, a significantly higher parasite load was detected using qPCR when DNA was extracted using Q versus SX. Considering the cost, execution time, feasibility, and performance of RPA assay, rapid extraction methods such as the Boil and Spin technique appear to have the potential for implementation in resource-limited endemic settings. Further clinical research is warranted prior to broader application.

Absolute quantification of nucleic acid on digital microfluidics platform based on superhydrophobic–superhydrophilic micropatterning

Digital microfluidics (DMF) shows great potential in the manipulation of individual droplets. However, the limited number of electrode control wirings for droplet control intrinsically restrains the massive generation of pL/nL individual droplets, fundamentally hindering the realization of high throughput applications on DMF such as digital nucleic acid amplification and analysis. This work demonstrates on-chip high throughput digital loop-mediated isothermal amplification (dLAMP) on a DMF platform based on a functional substrate with super-wettability contrast. Exploiting the passive dispensing technique as empowered by electrowetting on dielectric (EWOD) effect, a total of 1818 individual droplets with high uniformity are generated on the super-hydrophilic microarray within 1 min. Separation within the oil phase without tubes and valves can avoid evaporation, ensuring a precise total reaction volume. Absolute quantification of λDNA with a dynamic range of 1–1000 copies/μL is achieved by counting the amount of positive microdroplets after on-chip incubation, demonstrating the strong potential for rapid, low-cost, reliable, and quantitative nucleic acid analysis with high accuracy.

A hand-powered SERS-microfluidic chip for circulating tumor DNA detection from whole blood

Portable devices for rapid, automated and quantitative nucleic acid detection from whole blood is highly desirable for point-of-care (POC) molecular diagnostics. Conventional sequencing and polymerase chain reaction methods usually require trained personnel to deal with the time-consuming procedures, from sample purification to target amplification. To promote POC disease diagnosis and monitoring, we herein developed a hand-powered microfluidic chip for circulating tumor DNA (ctDNA) analysis from whole blood. On the one hand, the chip allowed rapid blood cell removal through a microfluidic filter trench. On the other hand, the ultrasensitivity of surface enhanced Raman spectroscopy (SERS) technique enabled amplification-free DNA mutation screening. Meanwhile, a finger-driven vacuum system was incorporated into the system, which achieved microfluidic pumping without external pumps, tubes or power sources. The developed SERS chip achieved fast screening of EGFR E746-A750 mutation in 35 min, reaching a limit of detection down to 100 fM in blood. Due to the simplicity, fast analysis, low cost and quantitative readout, the chip is expected to expand the availability of molecular diagnosis into resource-limited and bedside settings.

EE188 A Retrospective Cohort Comparing the Tuberculosis Diagnosis Related Costs and Health Resource Use (HRU) Using a Rapid Nucleic Acid Amplification (NAA) Test Vs. Conventional Tests in China, Followed By a Budget Impact Model

EE188 A Retrospective Cohort Comparing the Tuberculosis Diagnosis Related Costs and Health Resource Use (HRU) Using a Rapid Nucleic Acid Amplification (NAA) Test Vs. Conventional Tests in China, Followed By a Budget Impact Model

Development of a novel integrated isothermal amplification system for detection of bacteria-spiked blood samples

Bloodstream infection (BSI) caused by bacteria is highly pathogenic and lethal, and easily develops whole-body inflammatory state. Immediate identification of disease-causing bacteria can improve patient prognosis. Traditional testing methods are not only time-consuming, but such tests are limited to laboratories. Recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) holds great promise for rapid nucleic acid detection, but the uncapping operation after amplification easily contaminates laboratories. Therefore, the establishment of a more effective integrated isothermal amplification system has become an urgent problem to be solved. In this study, we designed and fabricated a hermetically sealed integrated isothermal amplification system. Combining with this system, a set of RPA-LFD assays for detecting S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI were established and evaluated. The whole process could be completed in less than 15 min and the results can be visualized by the naked eye. The developed RPA-LFD assays displayed a good sensitivity, and no cross-reactivity was observed in seven similar bacterial genera. The results obtained with 60 clinical samples indicated that the developed RPA-LFD assays had high specifcity and sensitivity for identifying S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI. In conclusion, our results showed that the developed RPA-LFD assay is an alternative to existing PCR-based methods for detection of S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI in primary hospitals.

Hydrogel Digital LAMP with Suppressed Nonspecific Amplification for Rapid Diagnostics of Fungal Disease in Fresh Fruits.

Fungal disease, mainly caused by Alternaria alternata infection, can generate severe economic losses and health hazards. However, rapid nucleic acid test without nonspecific reaction still remains challenging. Here, we reported the hydrogel digital loop-mediated isothermal amplification (HdLAMP) with suppressed nonspecific amplification for rapid diagnosis of fungi in fresh fruits. The introduction of hydrogel offered a simple platform to achieve absolute quantification. By breaking the 3'end G-C anchor, the nonspecific amplification of primers could be suppressed, while the specific positive reaction in HdLAMP was not affected. This method could be applied for A. alternata detection in 9 min with excellent performances in speed, specificity, reproducibility, sensitivity, and detection limit down to a single copy. Finally, the real diseased jujubes during postharvest storage were successfully diagnosed as an A. alternata infection. HdLAMP promotes the molecular diagnosis of fungal diseases and broadens the application of hydrogels in the agricultural and food industry.

Loop-mediated Iso-thermal amplification (LAMP) used for COVID-19 detection

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technique, which can be efficiently amplified at constant temperature by using a Bst DNA polymerase with high chain replacement activity. Compared with traditional nucleic acid amplification technology, this technique has the advantages of high sensitivity, strong specificity, simple operation and low detection cost. In the outbreak of COVID-19 in recent years, LAMP technology has been successfully used to develop rapid domestic COVID-19 nucleic acid detection kits with the above advantages, which can achieve rapid detection of COVID-19 in only 20min, greatly improving the convenience and immediacy of nucleic acid detection. In this paper, the principle and development of LAMP technology were summarized by using databases such as NCBI PubMed, Web of Science, CNKI, etc., focusing on the application of LAMP technology in novel coronavirus detection, and the future development direction of LAMP technology was analyzed by describing the latest LAMP technology.

Loop-mediated isothermal amplification of economically important Ceratitis species (Diptera: Tephritidae).

Ceratitis is an economically important genus of fruit flies that originated in Africa, has a wide host range, and causes serious economic losses due to its invasive damage. As a result, it is critical to identify them accurately and quickly in the world. Loop-mediated isothermal amplification (LAMP), as one of the representatives of isothermal amplification technology, has been widely used in the rapid nucleic acid detection of human pathogens and has shown its advantages in the identification of insect agricultural pests. In this study, using the mitochondrial cox1 and cob genes as target genes, the rapid molecular identification of the Ceratitis FARQ complex, C. cosyra, and C. capitata was realized based on LAMP. The experimental conditions optimization results showed that F3/B3:FIP/BIP = 1:8 was the optimal primer concentration ratio and 63 °C was the optimal reaction temperature. The sensitivity of the primers obtained in this study can reach up to 0.01 ng/μl DNA. A loop-mediated isothermal amplification identification technology system was established based on rapid, rough DNA extraction and visual detection of Ceratitis economically important fruit flies. The positive reaction system changed from pink to khaki by visual detection. The identification flow can be completed within 1 hour, including sample processing, DNA extraction, and LAMP visual detection.

Living Magnetotactic Microrobots Based on Bacteria with a Surface-Displayed CRISPR/Cas12a System for Penaeus Viruses Detection.

Bacterial microrobots are an emerging living material in the field of diagnostics. However, it is an important challenge to make bacterial microrobots with both controlled motility and specific functions. Herein, magnetically driven diagnostic bacterial microrobots are prepared by standardized and modular synthetic biology methods. To ensure mobility, the Mms6 protein is displayed on the surface of bacteria and is exploited for magnetic biomineralization. This gives the bacterial microrobot the ability to cruise flexibly and rapidly with a magnetization intensity up to about 18.65 emu g-1. To achieve the diagnostic function, the Cas12a protein is displayed on the bacterial surface and is used for aquatic pathogen nucleic acid detection. This allows the bacterial microrobot to achieve sensitive, rapid, and accurate on-site nucleic acid detection, with detection limits of 8 copies μL-1 for decapod iridescent virus 1 (DIV1) and 7 copies μL-1 for white spot syndrome virus (WSSV). In particular, the diagnostic results based on the bacterial microrobots remained consistent with the gold standard test results when tested on shrimp tissue. This approach is a flexible and customizable strategy for building bacterial microrobots, providing a reliable and versatile solution for the design of bacterial microrobots.

Integrated Microwell Array-Based Microfluidic Chip with a Hand-Held Smartphone-Controlled Device for Nucleic Acid Detection.

In this study, we designed a highly integrated microfluidic chip for nucleic acid extraction, amplification, and detection. Magnetic beads, which are used to capture nucleic acids on the chip, are trapped in the microwell arrays in a one-well-one-bead manner after local surface modification of the inner faces of the microwells. On-chip liquid introduction, delivery, and mixing are all carried out manually with one syringe and no other equipment. A hand-held device with precise temperature control and high-quality imaging is developed, which is only 2.3 cubic decimeters in volume and 1.2 kg in weight. Via the use of the Internet for wireless communication, the experiment and data analysis after inserting the chip into the device can be conducted by a smartphone anywhere there is an Internet connection. We carried out reverse transcription loop-mediated isothermal amplification (RT-LAMP) on the chip with the hand-held device. SARS-CoV-2 pseudoviruses are extracted, reverse transcribed, amplified, and detected on the chip with the hand-held device with satisfactory results. Thus, a highly integrated, easy-to-operate, and rapid nucleic acid detection microfluidic chip with a hand-held smartphone-controlled device is proposed, and this new platform for nucleic acid detection shows great potential for mobile point-of-care testing (POCT).

Rapid ultra-sensitive nucleic acid detection using plasmonic fiber-optic spectral combs and gold nanoparticle-tagged targets

Nucleic acid (NA) is a widely-used biomarker for viruses. Accurate quantification of NA can provide a reliable basis for point-of-care diagnosis and treatment. Here, we propose a tilted fiber Bragg grating (TFBG)-based plasmonic fiber-optic spectral comb for fast response and ultralow limit NA detection. The TFBG is coated with a gold film which enables excitation of surface plasmon resonance (SPR), and single-stranded probe NAs with known base sequences are assembled on the gold film. To enhance sensitivity of refractive index (RI) for sensing a chosen combination of probe and target NAs around the TFBG surface, gold nanoparticles (AuNPs) are bonded to the target NA molecules as “RI-labels”. The NA combination-induced aggregation of AuNPs induces significant spectral responses in the TFBG that would be below the detection threshold for the NAs in the absence of the AuNPs. The proposed TFBG-SPR NA sensor shows a fast response time of 30 s and an ultra-wide NA detection range from 1 × 10−18 mol/L to 1 × 10−7 mol/L. In the NA concentration range of 1 × 10−12 mol/L (1 pM) to 105 pM, an ultra-high sensitivity of 1.534 dB/lg(pM) is obtained. The sensor achieves an ultra-low limit of detection down to 1.0 × 10−18 mol/L (1 aM), which is more than an order of magnitude lower than the previous reports. The proposed sensor not only shows potentials in practical applications of NA detection, but also provides a new way for TFBG-SPR biochemical sensors to achieve higher RI sensitivity.

B-232 Versatile, Extraction-Free Nucleic Acid Sample Preparation in 15 Seconds Via Hyperbaric Heating

Abstract Nucleic acid extraction-based PCR sample prep is currently limited by long processing times, liquid transfers, kit cost, and lab storage requirements. Extraction-free approaches enable streamlined sample prep, but lack sensitivity compared to extraction-based methods. We studied several sample types to demonstrate the effectiveness of a novel 15-second Hyperbaric Heating (HBH) process to prepare nucleic acids for PCR. HBH exposes the sample to a short duration, high temperature environment to effectively lyse organisms and denature PCR inhibitors such as nucleases in seconds. Samples are introduced and sealed in a pressure-tight metallic vessel, which is then heated in seconds using magnetic induction. Within the sealed metallic vessel, the vaporization of the aqueous sample raises the internal pressure, enabling an internal temperature higher than 100°C to be achieved. Chelating agents and other proteins in HBH also protect the target nucleic acids from fragmentation. Post HBH, the sample is ready for direct PCR amplification without further dilution or extraction. Efficacy of HBH for efficient PCR amplification was tested against commercial kits. HBH was applied to viral, bacterial, eukaryotic, and fungal organisms. For SARS-CoV-2, HBH detected down to 250 viral copies/mL. Additionally, HBH outperformed commercial kits for PCR sample prep of Candida albicans and Bacillus subtilis. HBH is a generalizable sample prep technology with sensitivity comparable to that of current extraction kits. We have applied HBH to a multitude of organisms and have demonstrated that HBH can be employed as a rapid and comprehensive nucleic acid prep approach for applications that involve PCR amplification post sample prep. Future integration of HBH in devices suitable for point of care molecular testing are underway. We also envision HBH to be a useful, generalizable sample prep method for the molecular lab in applications where cost, simplicity, and speed are essential for preparing nucleic acids for amplification.

Advances in Cas12a-Based Amplification-Free Nucleic Acid Detection.

In biomedicine, rapid and sensitive nucleic acid detection technology plays an important role in the early detection of infectious diseases. However, most traditional nucleic acid detection methods require the amplification of nucleic acids, resulting in problems such as long detection time, complex operation, and false-positive results. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) systems have been widely used in nucleic acid detection, especially the CRISPR-Cas12a system, which can trans cleave single-stranded DNA and can realize the detection of DNA targets. But, amplification of nucleic acids is still required to further improve detection sensitivity, which makes Cas12a-based amplification-free nucleic acid detection methods a great challenge. This article reviews the recent progress of Cas12a-based amplification-free detection methods for nucleic acids. These detection methods apply electrochemical detection methods, fluorescence detection methods, noble metal nanomaterial detection methods, and lateral flow assay. Under various optimization strategies, unamplified nucleic acids have the same sensitivity as amplified nucleic acids. At the same time, the article discusses the advantages and disadvantages of each method and further discusses the current challenges such as off-target effects and the ability to achieve high-throughput detection. Amplification-free nucleic acid detection technology based on CRISPR-Cas12a has great potential in the biomedical field.

Development and application of a rapid scheme for detection of respiratory virus nucleic acid

This study aimed to develop a portable, accurate and easy-to-operate scheme for rapid detection of respiratory virus nucleic acid. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the effect of extraction-free respiratory virus treatment reagent (RTU) on viral nucleic acid treatment and the effect of ultra-fast fluorescence quantitative PCR instrument (FQ-8A) on nucleic acid amplification, respectively. RTU and FQ-8A were combined to develop a rapid detection scheme for respiratory virus nucleic acid, and the positive detection rate was judged by Ct value using a fluorescence quantitative PCR instrument, and the accuracy of the scheme in clinical samples detection was investigated. The results showed that RTU had comparable sensitivity to the automatic nucleic acid extraction instrument, its extraction efficiency was comparable to the other 3 extraction methods when extracting samples of different virus types, but the extraction time of RTU was less than 5 min. FQ-8A had good consistency in detection respiratory syncytial virus (RSV) and adenovirus (ADV) compared with the control instrument ABI-7500, with kappa coefficients of 0.938 (P < 0.001) and 0.887 (P < 0.001), respectively, but the amplification time was only about 0.5 h. The RTU and FQ-8A combined rapid detection scheme had a highly consistent detection rate with the conventional detection scheme, with a sensitivity of 91.70% and specificity of 100%, and a kappa coefficient was 0.944 (P < 0.001). In conclusion, by combining RTU with FQ-8A, a rapid respiratory virus nucleic acid detection scheme was developed, the whole process could be completed in 35 min. The scheme is accurate and easy-to-operate, and can provide important support for the rapid diagnosis and treatment of respiratory virus.

Scanning Electron Microscopy and EDX Spectroscopy of Commercial Swabs Used for COVID-19 Lateral Flow Testing.

The chemical composition of COVID test swabs has not been examined beyond the manufacturer's datasheets. The unprecedented demand for swabs to conduct rapid lateral flow tests and nucleic acid amplification tests led to mass production, including 3D printing platforms. Manufacturing impurities could be present in the swabs and, if so, could pose a risk to human health. We used scanning electron microscopy and energy dispersive X-ray (EDX) spectroscopy to examine the ultrastructure of seven assorted brands of COVID test swabs and to identify and quantify their chemical elements. We detected eight unexpected elements, including transition metals, such as titanium and zirconium, the metalloid silicon, as well as post-transition metals aluminium and gallium, and the non-metal elements sulphur and fluorine. Some of the elements were detected as trace amounts, but for others, the amount was close to reported toxicological thresholds for inhalation routes. Experimental studies have shown that the detrimental effects of unexpected chemical elements include moderate to severe inflammatory states in the exposed epithelium as well as proliferative changes. Given the massive testing still being used in the context of the COVID pandemic, we urge caution in continuing to recommend repeated and frequent testing, particularly of healthy, non-symptomatic, individuals.

Research on a Magnetic Separation-Based Rapid Nucleic Acid Extraction System and Its Detection Applications.

Nucleic acid extraction represents the "first step" in molecular diagnostic experiments. The quality of this extraction serves as a fundamental prerequisite for ensuring the accuracy of nucleic acid detection. This article presents a comprehensive design scheme for a rapid automated nucleic acid extraction system based on magnetic separation. The design and implementation of the system are analyzed and investigated in-depth, focusing on the core methods, hardware control, and software control of the automated nucleic acid extraction system. Additionally, a study and evaluation were carried out concerning the nucleic acid extraction and detection aspects encompassed by the system. The results demonstrate that the temperature deviation in the lysis and elution fluids is approximately ±1 °C, the positioning accuracy of the system's movement is ±0.005 mm, the average magnetic bead recovery rate is 94.98%, and the average nucleic acid recovery rate is 91.83%. The developed automated system and manual methods are employed for sample extraction, enabling the isolation of highly pure nucleic acids from bacteria, blood, and animal tissues for RT-PCR detection. The instrument employs lysis temperatures ranging from 70-80 °C, elution temperature of 80 °C, and drying time of 5-10 min, with a total extraction time of less than 35 min for different sample types. Overall, the system yields high nucleic acid concentration and purity, exhibits stable instrument operation, good repeatability, high efficiency, and low cost. It meets the requirements of genetic-level research and is worthy of clinical promotion and usage.

Simple Point-of-Care Nucleic Acid Amplification Test for Rapid SARS-CoV-2 Infection Diagnosis.

After three years of the SARS-CoV-2 pandemic, the demand for developing field-deployable point-of-care (PoC) molecular diagnostic tests has increased. Although RT-qPCR is the molecular diagnostic gold standard and is accurate, it is not readily applied to point-of-care testing (POCT). Meanwhile, rapid diagnostic kits have the disadvantage of low sensitivity. Recently, rapid isothermal nucleic acid amplification technology has emerged as an alternative for rapid diagnosis. Here, we developed a rapid SARS-CoV-2 reverse transcription loop-mediated isothermal amplification (RT-LAMP)-lateral flow assay (LFA) kit. This kit includes a Chelex-100/boiling nucleic acid extraction device and a one-step amplification detection apparatus capable of performing the entire process, from RNA extraction to detection, and diagnosing SARS-CoV-2 infection within 40 min without contamination. The detection limits of the rapid SARS-CoV-2 RT-LAMP-LFA kit were 100 plaque-forming units (PFUs) mL-1 and 10-1 PFU mL-1 for RNA samples extracted using the Chelex-100/boiling nucleic acid extraction device and commercial AdvansureTM E3 system, respectively. The sensitivity and specificity of the rapid SARS-CoV-2 RT-LAMP-LFA kit were 97.8% and 100%, respectively. Our SARS-CoV-2 RT-LAMP-LFA kit exhibited high sensitivity and specificity within 40 min without requiring laboratory instruments, suggesting that the kit could be used as a rapid POC molecular diagnostic test for SARS-CoV-2.

Deep Learning Assisted Surface-Enhanced Raman Spectroscopy (SERS) for Rapid and Direct Nucleic Acid Amplification and Detection: Toward Enhanced Molecular Diagnostics.

Surface-enhanced Raman scattering (SERS) has evolved into a robust analytical technique capable of detecting a variety of biomolecules despite challenges in securing a reliable Raman signal. Conventional SERS-based nucleic acid detection relies on hybridization assays, but reproducibility and signal strength issues have hindered research on directly amplifying nucleic acids on SERS surfaces. This study introduces a deep learning assisted ZnO-Au-SERS-based direct amplification (ZADA) system for rapid, sensitive molecular diagnostics. The system employs a SERS substrate fabricated by depositing gold on uniformly grown ZnO nanorods. These nanorods create hot spots for the amplification of the target nucleic acids directly on the SERS surface, eliminating the need for postamplification hybridization and Raman reporters. The limit of detection of the ZADA system was superior to those of the conventional amplification methods. Clinical validation of the ZADA system with coronavirus disease 2019 (COVID-19) samples from human patients yielded a sensitivity and specificity of 92.31% and 81.25%, respectively. The integration of a deep learning program further enhanced sensitivity and specificity to 100% and reduced SERS analysis time, showcasing the potential of the ZADA system for rapid, label-free disease diagnosis via direct nucleic acid amplification and detection within 20 min.

Argonaute protein-based nucleic acid detection technology.

It is vital to diagnose pathogens quickly and effectively in the research and treatment of disease. Argonaute (Ago) proteins are recently discovered nucleases with nucleic acid shearing activity that exhibit specific recognition properties beyond CRISPR-Cas nucleases, which are highly researched but restricted PAM sequence recognition. Therefore, research on Ago protein-mediated nucleic acid detection technology has attracted significant attention from researchers in recent years. Using Ago proteins in developing nucleic acid detection platforms can enable efficient, convenient, and rapid nucleic acid detection and pathogen diagnosis, which is of great importance for human life and health and technological development. In this article, we introduce the structure and function of Argonaute proteins and discuss the latest advances in their use in nucleic acid detection.

Rapid and portable bunyavirus SFTSV RNA testing utilizing catalytic hairpin assembly coupled with lateral flow immunoassay.

Severe fever with thrombocytopenia syndrome (SFTS) is a prevalent, life-threatening, emergent infectious disease. Currently, reverse transcription-polymerase chain reaction is the gold standard for diagnosing SFTS virus (SFTSV) infection, which requires sophisticated equipment and professional personnel that are frequently unavailable in most SFTS endemic rural areas. Here, we reported a simple, rapid nucleic acid amplification system that combined the catalytic hairpin assembly (CHA) with a lateral flow immunoassay (LFIA) strip-based detection method for SFTSV detection. The detection of SFTSV RNA could be realized by generation of H1-H2 hybrid duplexes labeled with biotin and digoxin, which subsequently added to the LFIA test strips containing streptavidin conjugated with Alexa Fluor 647 as well as anti-digoxin antibodies. Our CHA-based LFIA assay offered high amplification efficiency and specificity with a detection limit of 1 aM. Crucially, this method enabled stable detection of 500 copies/mL of SFTSV within 30 min using clinical serum samples. Therefore, our CHA-based LFIA approach provided a potential useful tool to facilitate early and precise diagnosis of SFTS patients in poorly resourced SFTS endemic areas.IMPORTANCESevere fever with thrombocytopenia syndrome (SFTS) is an emerging and potentially fatal infectious disease prevalent in China. Here we report a simple, rapid nucleic acid amplification system, the catalytic hairpin assembly (CHA) in conjunction with a lateral flow immunoassay (LFIA) strip-based detection method for SFTS virus detection, which demonstrated high amplification efficiency and specificity with limit of detection of 1 aM. Most importantly, we also validate our CHA-based LFIA assay using the clinical serum samples, which was fully compatible with reverse transcription-PCR results. Therefore, our strategy provides a potential useful tool to facilitate early and precise diagnosis of SFTS patients especially in poorly resourced SFTS endemic areas.