ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Kai Wu,Qi Fang,Zhantao Zhao,Zida Li
Multiplex, digital nucleic acid tests have important biomedical applications, but existing methods mostly use fluorescent probes that are target-specific and difficult to optimize, limiting their widespread applications. Here, we report color-encoded, intelligent digital loop-mediated isothermal amplification (CoID-LAMP) for the coidentification of multiple nucleic acid targets. CoID-LAMP supplements different primer solutions with different dyes, generates primer droplets and sample droplets, and collectively pairs these two types of droplets in a microwell array device to perform LAMP. After imaging, the droplet colors were analyzed to decode the primer information, and the precipitate byproducts within droplets were detected to determine the target occupancy and calculate the concentrations. We first established an image analysis pipeline based on a deep learning algorithm for reliable droplet detection and validated the analytical performance in nucleic acid quantification. We then implemented CoID-LAMP using fluorescent dyes as the coding materials and established an 8-plex digital nucleic acid assay, confirming the reliable coding performance and the capability of multiplex nucleic acid quantification. We further implemented CoID-LAMP using brightfield dyes for a 4-plex assay, suggesting that the assay could be realized solely by brightfield imaging with minimal demand on the optics. Leveraging the advantages of droplet microfluidics in multiplexing and deep learning in intelligent image analysis, CoID-LAMP offers a useful tool for multiplex nucleic acid quantification.
研究领域:
生物医学工程
·
分子生物学
·
分析化学
·
微流控技术
·
机器学习
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Virology Journal
·
2025
·
IF 3.8
Jinlei Guo,Yunfeng Shan,Ge Hu,Xiaojuan Zhu,Kangchen Zhao,Tao Wu,Qiao Qiao,Ying Chi,Lunbiao Cui,Yiyue Ge
Background Monkeypox virus (MPXV) infection has garnered significant global attention due to its rising incidence and substantial public health implications. A rapid, sensitive, and accurate diagnostic method is urgently required to enable early intervention and effective management of MPXV outbreaks. Results In this study, we developed a novel one-step assay that integrates loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12b in one-pot for the detection of MPXV. The entire detection process did not require opening the lid of the reaction tube and could be completed within 40 min using extracted viral nucleic acids, which is faster than real-time quantitative PCR (qPCR). And the results could be interpreted through either real-time fluorescence or naked-eye visualization. The limit of detection (LOD) of the assay was demonstrated to be 6.5 copies per reaction and no cross-reactivity with other pathogens such as HSV, EBV, CVA16, EV-A71, and MV was found. Furthermore, when evaluated using 113 clinical samples, the assay achieved 100% sensitivity (71/71) and 100% specificity (42/42) compared to the qPCR. Conclusions In resource-limited settings, our method requires only a portable heat block or water bath and a blue light or ultraviolet flashlight for visual detection of MPXV, making it highly accessible. The integration of LAMP and CRISPR/Cas12b provides a robust, user-friendly platform for point-of-care testing, with promising potential for the rapid molecular diagnosis of infectious diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s12985-025-02780-0.
研究领域:
医学
·
分子生物学
·
传染病学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Science Advances
·
2024
·Vol. 10
·期 46
·
IF 11.7
Wenshang Guo,Ye Tao,Ruizhe Yang,Kaihao Mao,Hongwei Zhou,Minghui Xu,Tie Sun,Xiao Li,Changrui Shi,Zhenyou Ge,Rui Xue,Haizhou Zhou,Yukun Ren
Developing instant detection systems with disease diagnostic capabilities holds immense importance for remote or resource-limited areas. However, the task of creating these systems—which are simultaneously easy to operate, rapid in detection, and cost-effective—remains a challenge. In this study, we present a compact highly sensitive photothermal reverse transcriptase–loop-mediated isothermal amplification (RT-LAMP) chip (SPRC) designed for the detection of multiple diseases. The nucleic acid (NA) amplification on the chip is achieved through LAMP driven by either LED illumination or simple sunlight focusing. SPRC performs sample addition and amplification within a limited volume and autonomous enrichment of NA during the sample addition process, achieving a limit of detection (LOD) as low as 0.2 copies per microliter. Through 120 clinical samples, we achieved an accuracy of 95%, with a specificity exceeding 97.5%. Overall, SPRC has achieved promising progress in the application of point-of-care testing (POCT) by using light energy to simultaneously detect multiple diseases.
研究领域:
分子生物学
·
医学检验
·
生物医学工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Scientific Reports
·
2014
·Vol. 4
·期 1
·1-8
·
IF 5.078
Zhou Dinggang,Guo Jinlong,Xu Liping,Gao Shiwu,Lin Qingliang,Wu Qibin,Wu Luguang,Que Youxiong
To meet the demand for detection of foreign genes in genetically modified (GM) sugarcane necessary for regulation of gene technology, an efficient method with high specificity and rapidity was developed for the cry1Ac gene, based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed using the sequence of cry1Ac along with optimized reaction conditions: 5.25 mM of Mg 2+ , 4:1 ratio of inner primer to outer primer, 2.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. Three post-LAMP detection methods (precipitation, calcein (0.60 mM) with Mn 2+ (0.05 mM) complex and SYBR Green I visualization), were shown to be effective. The sensitivity of the LAMP method was tenfold higher than that of conventional PCR when using templates of the recombinant cry1Ac plasmid or genomic DNA from cry1Ac transgenic sugarcane plants. More importantly, this system allowed detection of the foreign gene on-site when screening GM sugarcane without complex and expensive instruments, using the naked eye. This method can not only provide technological support for detection of cry1Ac , but can also further facilitate the use of this detection technique for other transgenes in GM sugarcane.
研究领域:
分子生物学
·
植物生物技术
·
基因工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Frontiers in Cellular and Infection Microbiology
·
2025
·
IF 4.8
Yizhuo Zhang,Luqin Lv,Su Xu,Yijian Chen
Introduction: Clostridioides difficile (C. difficile), a human pathogen that causes diarrhea and colon lesions, has garnered widespread attention. Rapid and accurate detection of bacterial virulence factors is essential for the diagnosis of C. difficile infection (CDI). To date, numerous laboratory tests have been developed; however, none fully meet the combined requirements of speed, cost-effectiveness, portability, sensitivity, and specificity. Molecular diagnostic technologies based on CRISPR-Cas systems have provided a promising solution to this challenge. Nonetheless, the limited compatibility between pre-amplification and CRISPR cleavage, coupled with the inherent selectivity of CRISPR systems for protospacer adjacent motif (PAM) sequences near the target site, poses additional constraints on the broader adoption of this approach.Methods: Here, we developed PAM-unconventional, One-step LAMP/CRISPR-Cas12b (POLC) detection platforms for the toxin-encoding genes tcdA and tcdB of C. difficile.Results: The POLC platforms operated at 60 °C, enabling result interpretation either through fluorescence intensity measurements or direct visualization under UV light. The limits of detection (LoDs) ranged from 3 to 14 copies/μL using a fluorescence reader and from 6 to 18 copies/μL via direct observation. Compared to qPCR, which typically requires over an hour, the POLC platforms reduced the detection time to approximately 40 minutes. Each reaction cost approximately USD 6.5, offering a substantial cost saving compared to qPCR-based commercial kits (over USD 10 per test). In clinical validation with 55 fecal samples, the tcdA POLC assay achieved 86.4% sensitivity and 84.8% specificity, while the tcdB POLC assay demonstrated 96.6% sensitivity and 100% specificity, using qPCR as the reference standard.Discussion: Our research presents innovative CRISPR-based one-step nucleic acid detection platforms that eliminate canonical PAM sequence requirements. These platforms exhibit high sensitivity and specificity while achieving rapid detection under simple conditions, making them promising candidates for clinical diagnostics and point-of-care testing (POCT).
研究领域:
医学诊断
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
LWT-FOOD SCIENCE AND TECHNOLOGY
·
2023
·
IF 6
Yingchun Li,Yaqin Zhang,Fange Kong,Chunxia Wang,Shanshan Chen,Jiasi Wang,Di Wang
Fusarium temperatum ( F. temperatum ) causes maize stalk rot disease, reducing grain yield and producing multiple harmful mycotoxins that threaten food safety and quality. However, current detection methods are limited by the requirement of well-trained personnel, complicated operations and expensive instruments. To address this challenge, gold nanoparticles (AuNPs)-based colorimetric assay using loop-mediated isothermal amplification (LAMP) coupled with CRISPR-Cas12a (Au-CRISPR) was tested for F. temperatum detection. The assay utilizes DNA-modified AuNPs as a colorimetric probe and the results can be readily observed by the naked eye. After optimization, the method can specifically detect as low as 100 copies/μL of target DNA or 10 −8 ng/μL of extracted DNA from F. temperatum . The detection platform allows high-throughput testing by integrating microtiter plates and microplate reader. Furthermore, a portable smartphone-based device was developed to realize point of care (POC) detection of F. temperatum in resource-limited settings. This simple, inexpensive and sensitive detection platform has great potential for the applications in field detection.
研究领域:
植物病理学
·
分子生物学
·
食品安全
✓ 已验证
产品关联
DOI
高影响因子
PLANT DISEASE
·
2021
·
IF 4.438
Ran Du,Yongju Huang,Jing Zhang,Long Yang,Mingde Wu,Guo-qing Li
Blackleg of oilseed rape is a damaging invasive disease caused by the species complex Leptosphaeria maculans (Lm)/ L . biglobosa (Lb), which is composed of at least two and seven phylogenetic subclades, respectively. Generally, Lm is more virulent than Lb, but under certain conditions, Lb can cause a significant yield loss in oilseed rape. Lb ‘brassicae’ (Lbb) has been found to be the causal agent for blackleg of oilseed rape in China, whereas Lm and Lb ‘canadensis’ (Lbc) were frequently detected in imported seeds of oilseed rape, posing a risk of spread into China. To monitor the blackleg-pathogen populations, a diagnostic tool based on loop-mediated isothermal amplification (LAMP) was developed using a 615-bp–long DNA sequence from Lbb that was derived from a randomly amplified polymorphic DNA assay. The LAMP was optimized for temperature and time, and tested for specificity and sensitivity using the DNA extracted from Lbb, Lbc, Lm, and 10 other fungi. The results showed that the optimal temperature and time were 65°C and 40 min, respectively. The LAMP primer set was specific to Lbb and highly sensitive as it detected the Lbb DNA as low as 132 fg per reaction. The LAMP assay was validated using the DNA extracted from mycelia and conidia of a well-characterized Lbb isolate, and its utility was evaluated using the DNA extracted from leaves, stems, pods, and seeds of oilseed rape. The LAMP assay developed herein will help for monitoring populations of the blackleg pathogens in China and in developing strategies for management of the blackleg disease.
研究领域:
植物病理学
·
分子生物学
·
农业科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Biosensors-Basel
·
2023
·Vol. 13
·期 1
·111
·
IF 5.4
Tianyi Yang,Yanju Chen,Jinsong He,Jian Wu,Meixia Wang,Xiaoping Zhong
A rapid and intuitive method for detecting Vibrio parahaemolyticus (VP) was established by a designed reaction vessel which coupled CRISPR/Cas12a with loop-mediated isothermal nucleic acid amplification (LAMP). There were two spaces in the vessel-holding LAMP reaction solution and CRISPR reaction solution, respectively, which were separated with a polyvinyl alcohol (PVA) membrane. The PVA membrane could be dissolved with a water solution. The thermolabile hemolysin (TLH) gene of VP was employed as the detection target. After the target sequence of the TLH gene was amplified with LAMP, the PVA membrane would be dissolved and the CRISPR reaction solution mixed with the LAMP reaction solution. In this way, amplicons could be detected with CRISPR/Cas12a in the reaction vessel. The fluorescent signals produced by the positive samples were clearly identified by the naked eye under a UV light, while the negative samples were dark. The whole detection procedure could be finished within 35 min with a detection limit of 100 copies/µL. The designed reaction vessel is easy to produce and can effectively prevent contamination due to the opening of the reaction vessel after the LAMP reaction. Thus, it will have the potential to provide a new solution for rapid detection in the field.Keywords:polyvinyl alcohol;dissolvable membrane;CRISPR/Cas12a;LAMP reaction;Vibrio parahaemolyticus
研究领域:
分子生物学
·
病原体检测
·
生物技术
✓ 已验证
DOI
PubMed
高影响因子
PLANT DISEASE
·
2019
·Vol. 103
·期 12
·
IF 3.583
Q. W. Wang,C. Q. Zhang
Trunk canker disease caused by Botryosphaeria dothidea with a prolonged latent infection phase poses a serious threat to Chinese hickory production. To further understand the epidemiological characteristics and develop reasonable management techniques, a quantitative loop-mediated isothermal amplification (q-LAMP) assay was developed to quantitatively monitor B. dothidea in hickory plants, water, and air samples. Specific primers were designed based on the different sites of the β-tubulin sequence between B. dothidea and other fungi commonly found on Chinese hickory. At the optimum reaction temperature of 65.9°C, this loop-mediated isothermal amplification (LAMP) assay can specifically distinguish B. dothidea from other tested fungi. The limit of detection of LAMP assays for B. dothidea was 0.001 ng/µl of pure genomic DNA and 10 spores per 1 ml of water. The q-LAMP assay enables rapid detection of B. dothidea within 60 min in hickory trunk, water in hickory forests, and spores captured on tapes. These results provide a powerful and convenient tool for monitoring B. dothidea , which could be applied widely in epidemiology, forecast, and management of tree canker disease.
研究领域:
植物病理学
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2020
·
IF 7.1
Hui Kong,Wei Zhang,Jia Yao,Chao Li,Renfei Lu,Zhen Guo,Jinze Li,Chuanyu Li,Yingxue Li,Chiyu Zhang,Lianqun Zhou
Development of a simple, sensitive and specific method for HIV-1 assay is demanded. In some emergency situations, a portable reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform is necessary. A novel hydrogen ion-selective RT-LAMP (H + -RT-LAMP) sensor was developed for highly sensitive HIV-1 using hydrogen ion concentration as a signal producing compound. The H + -RT-LAMP sensor was based on screen-printed with carbon-doped graphene conductive layer, carbon nanotubes (CNTs) solid electrolyte layer, and reference electrode KCl functional layer, which provided superior electrical signals, high sensitivity and stability for HIV-1 detection. The rate of hydrogen ion production was related to the viral load (VL) during the amplification of HIV-1. The sensitive HIV-1 RNA detection range was achieved from 0 copies/μL to 10 5 copies/μL. The proposed H + -RT-LAMP sensor was applied to detect HIV-1 RNA in real human serum. 57 samples were detected and satisfactory results were obtained. Compared with commercial instruments, the detection results of H + -RT-LAMP sensor demonstrated a perfect concordance rate of 96.5 %, and the limit of detection (LOD) for the clinical samples was 10 copies per tube. Time to get the result is from 10 min to 25 min. Therefore, the proposed H + -RT-LAMP sensor was particularly suitable as a point-of-care device for the on-site detection of HIV-1.
研究领域:
分子生物学
·
医学检验
·
生物传感器
✓ 已验证
产品关联
DOI
高影响因子
FOOD CHEMISTRY
·
2023
·
IF 8.8
Bin Xiao,Ruiming Zhao,Nan Wang,Juan Zhang,Xiaoyun Sun,Fengchun Huang,Ailiang Chen
Most microfluidic-based “sample-in-result-out” systems suffer sophisticated microfluidic production processes, high-cost chips, and expensive instruments. They cannot be used in the meat market as well as farmer's markets in rural areas. Here, we developed a hand-held microfluidic chip system for on-site meat species qualitative authentication detection which integrated a simple microneedle DNA extraction and a visual loop-mediated isothermal amplification (LAMP). The chip can be used by easily pricking meat samples, simply hand-shaking the chip, and readily available isothermal heating instead of a complicated DNA extraction process and microfluidic control device. The system demonstrates high specificity and sensitivity for selected six species of meat samples and low to 1% simulated adulteration could be detected within 60 min. Besides, the whole cost was less than 1 dollar. The integrated hand-held microfluidic detection system offers a simple, fast, low-cost “sample-in-result-out” point-of-care device which could be extended to medical diagnosis and animal/plant disease identification.
研究领域:
生物技术
·
食品科学
·
分子生物学
·
即时诊断
✓ 已验证
产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2021
·
IF 7.46
Yaqin Zhang,Minyan Chen,Chengrong Liu,Jiaqi Chen,Xinyi Luo,Yingying Xue,Qiming Liang,Li Zhou,Yu Tao,Mingqiang Li,Di Wang,Jianhua Zhou,Jiasi Wang
The outbreak of corona virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic. The high infectivity of SARS-CoV-2 highlights the need for sensitive, rapid and on-site diagnostic assays of SARS-CoV-2 with high-throughput testing capability for large-scale population screening. The current detection methods in clinical application need to operate in centralized labs. Though some on-site detection methods have been developed, few tests could be performed for high-throughput analysis. We here developed a gold nanoparticle-based visual assay that combines with CRISPR/Cas12a-assisted RT-LAMP, which is called C as12a-assisted RT- LA MP/AuN P (CLAP) assay for rapid and sensitive detection of SARS-CoV-2. In optimal condition, we could detect down to 4 copies/μL of SARS-CoV-2 RNA in 40 min. by naked eye. The sequence-specific recognition character of CRISPR/Cas12a enables CLAP a superior specificity. More importantly, the CLAP is easy for operation that can be extended to high-throughput test by using a common microplate reader. The CLAP assay holds a great potential to be applied in airports, railway stations, or low-resource settings for screening of suspected people. To the best of our knowledge, this is the first AuNP-based colorimetric assay coupled with Cas12 and RT-LAMP for on-site diagnosis of COVID-19. We expect CLAP assay will improve the current COVID-19 screening efforts, and make contribution for control and mitigation of the pandemic.
研究领域:
分子生物学
·
病毒学
·
诊断检测
·
纳米技术
·
公共卫生
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）