Frontiers in Cellular and Infection Microbiology
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2025
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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
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分子生物学
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DOI
PubMed
高影响因子
ARCHIVES OF TOXICOLOGY
·
2022
·Vol. 97
·期 2
·441-456
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IF 6.168
Xu Chi,Jiang Shuyun,Ma Xingyu,Jiang Zeyong,Pan Youguang,Li Xiang,Zhang Liying,Zhou Hao,Chen Shen,Xing Xiumei,Chen Liping,Fu Wenfan,Wang Qing,Chen Wen,Li Daochuan
Cisplatin is recommended as a first-line chemotherapeutic agent against advanced non-small cell lung cancer (NSCLC), but acquired resistance substantially limits its clinical efficacy. Recently, DNA methylation has been identified as an essential contributor to chemoresistance. However, the precise DNA methylation regulatory mechanism of cisplatin resistance remains unclear. Here, we found that nicotinamide nucleotide transhydrogenase ( NNT ) was silenced by DNA hypermethylation in cisplatin resistance A549 (A549/DDP) cells. Also, the DNA hypermethylation of NNT was positively correlated to poor prognosis in NSCLC patients. Overexpression of NNT in A549/DDP cells could reduce their cisplatin resistance, and also suppressed their tumor malignancy such as cell proliferation and clone formation. However, NNT enhanced sensitivity of A549/DDP cells to cisplatin had little to do with its function in mediating NADPH and ROS level, but was mainly because NNT could inhibit protective autophagy in A549/DDP cells. Further investigation revealed that NNT could decrease NAD + level, thereby inactivate SIRT1 and block the autophagy pathway, while re-activation of SIRT1 through NAD + precursor supplementation could antagonize this effect. In addition, targeted demethylation of NNT CpG island via CRISPR/dCas9-Tet1 system significantly reduced its DNA methylation level and inhibited the autophagy and cisplatin resistance in A549/DDP cells. Thus, our study found a novel chemoresistance target gene NNT , which played important roles in cisplatin resistance of lung cancer cells. Our findings also suggested that CRISPR-based DNA methylation editing of NNT could be a potential therapeutics method in cisplatin resistance of lung cancer.
研究领域:
肿瘤学
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分子生物学
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表观遗传学
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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
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分子生物学
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食品安全
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DOI
高影响因子
ANALYST
·
2025
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IF 3.3
Chenyan Li,Yilan Liang,Qincong Feng
Electrochemical biosensors are frequently employed to identify harmful microbes and disease indicators. However, their practical applicability is constrained by poor signal amplification efficiency and immobilization processes on the probe surface. To get over these restrictions, we here integrated roll-cycling amplification (RCA) and CRISPR/Cas12a gene editing tools with electrochemical biosensors. We constructed an electrochemical biosensor based on RCA to activate the cleavage activity of Cas12a. First, by double-stranded nucleic acid aptamer (ds Apt) specifically binding to E. coli-2571, Apt-b was competitively isolated to bind to T4 ligase and produce circular DNA. This in turn activates RCA, which in turn activates the accessory cleavage activity of CRISPR/Cas12a, which in turn cleaves DNA sequences loaded onto the electrode, changing electrochemical signals. With a linear range of 1 × 102–1 × 107 CFU mL−1, a detection limit of 5.28 CFU mL−1, and experimental recoveries of 93.01–101.53%, the measured electrochemical signals were positively connected with the concentration of E. coli-2571 under the optimal experimental conditions. Therefore, by combining two approaches—RCA and CRISPR/Cas12a—our electrochemical biosensor was able to detect E. coli-2571 specifically and sensitively, providing new research opportunities for the detection of other harmful bacteria.
研究领域:
分子生物学
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生物传感技术
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微生物检测
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
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2024
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IF 6.7
Luyu Wei,Zhilong Wang,Yongzhen Dong,Deyang Yu,Yiping Chen
CRISPR/Cas12a fluorimetry has been extensively developed in the biosensing arena, on account of its high selectivity, simplicity, and rapidness. However, typical CRISPR/Cas12a fluorimetry suffers from low sensitivity due to the limited trans-cleavage efficiency of Cas12a, necessitating the integration of other preamplification techniques. Herein, we develop an enhanced CRISPR/Cas12a fluorimetry via a DNAzyme-embedded framework nucleic acid (FNAzyme) substrate, which was designed by embedding four CLICK-17 DNAzymes into a rigid tetrahedral scaffold. FNAzyme can not only enhance the trans-cleavage efficiency of CRISPR/Cas12a by facilitating the exposure of trans-substrate to Cas12a but also result in an exceptionally high signal-to-noise ratio by mediating enzymatic click reaction. Combined with a functional nucleic acid recognition module, this method can profile methicillin-resistant Staphylococcus aureus as low as 18 CFU/mL, whose sensitivity is approximately 54-fold higher than that of TaqMan probe-mediated CRISPR/Cas12a fluorimetry. Meanwhile, the method exhibited satisfactory recoveries in food matrices ranging from 80% to 101%. The DNA extraction- and preamplification-free detection format as well as the potent detection performance highlight its tremendous potential as a next-generation analysis tool.
研究领域:
生物传感
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分子诊断
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合成生物学
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DOI
PubMed
高影响因子
FlatChem
·
2024
·
IF 5.9
Taojian Fan,Shaohui Zhang,Changle Meng,Lingfeng Gao,Li Yan,Hao Wang,Xin Shi,Yanqi Ge,Han Zhang,Junqing Hu
Ultra-sensitive nucleic acid detection is important for rapid prevention of infectious diseases. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems with great target specificity and programmability have demonstrated amazing capabilities in the field of nucleic acid detection. However, most CRISPR/Cas systems-based strategies still rely on pre-amplification of nucleic acid, which limits their clinical application in point of care detection. Here, we designed a novel electrochemical CRISPR/Cas biosensor (called E-Sb CRISPR) that utilizes antimonene nanosheets (Sb NSs) modifications. Due to the strong interaction between Sb NSs and single stranded DNA (ssDNA), E-Sb CRISPR exhibited specific nucleic acid detection capabilities with a detection limit of 100 aM within 35 min. Notably, the sensor showed excellent selectivity to target DNA in serum in the presence of nucleus acid extracted from other viruses. The excellent stability (8 weeks) and reproducibility (50 cycles) of this sensor were observed. By developing microcircuits and software systems, rapid acquisition of detection results on mobile electronic devices is possible. Therefore, the E-Sb CRISPR showed great promise as a scalable nucleic acid detection platform for early diagnosis.
研究领域:
分子生物学
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医学诊断
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生物传感技术
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DOI
高影响因子
MICROCHEMICAL JOURNAL
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2024
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IF 4.9
Yang Song,Yiwen Xu,Ruirui Wang,Jianping Wang,Xiaoping Yu,Xuping Shentu
Pathogenic bacterial contamination of food is a major challenge in food safety and public health management. To monitor pathogenic bacteria in food, a rapid, sensitive, and specific pathogen detection method is needed. CRISPR-Cas12a has been developed as a sensitive method for detecting pathogenic bacteria, but its output signal is fluorescent, which need equipment to detect. Here, the nanoparticle probe was designed to detect the activity of Cas12a and were able to visualize the assay results by observing the color change by naked eyes. The target sequences of pathogenic bacteria were amplified by recombinase polymerase, then recognized and bound by the crRNA and Cas12a complex. The activated Cas12a cleaved the linker-ssDNA connecting the nanoparticles, causing the nanoparticles to transition from aggregation to dispersion, and the color showed as purple. The sensitivity analysis showed that the limits of detection (LOD) reached 1 × 10°CFU/mL, 1 × 10 4 CFU/mL, 1 × 10 2 CFU/mL, 1 × 10 3 CFU/mL, 1 × 10 1 CFU/mL for Escherichia coli , Salmonella typhimurium , Vibrio parahaemolyticus , Staphylococcus aureus , and Listeria monocytogenes , which were all specific for five bacteria in the milk samples. Therefore, this method is a dependable, fast, sensitive and specific method, which make the CRIPSR-Cas12a detection method more useful in bacterial pathogenic detection.
研究领域:
食品科学
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分子生物学
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生物技术
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DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2022
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IF 9.221
Yu Tao,Ke Yi,Haixia Wang,Kai Li,Mingqiang Li
Since hepatitis B virus (HBV) infection can cause chronic viral hepatitis, increase the risk of liver fibrosis and cirrhosis, and ultimately result in hepatoma, it is of great significance to explore convenient, rapid, and sensitive diagnostic strategies for HBV DNA detection. In this study, we developed a novel and sensitive fluorescent biosensor for HBV detection based on the sensing system consisting of CRISPR-Cas12a enzymes and metal nanoclusters as the luminescent nanoprobes, involving gold nanoclusters (AuNCs), silver nanoclusters (AgNCs), and copper nanoclusters (CuNCs). The DNA probes could effectively be applied as the template for the preparation of fluorescent metal nanoclusters. In the presence of the HBV target, the trans-cleavage capability of Cas12a was able to be initiated to degrade DNA probes, which brought about the inhibition of metal nanoclusters formation, thereby giving minimal fluorescence signals. In comparison with AuNCs and AgNCs, the CuNCs-based nanosensor exhibited much higher sensitivity. In addition, the fluorescence response of CuNCs to HBV detection was significantly faster, which could be completed within 25 min without the requirements of sophisticated equipment or harsh reaction conditions. Moreover, the practical application of this analysis method was also demonstrated to detect HBV DNA target in human serum samples with satisfactory recovery, which was promising for simple, rapid, sensitive, and label-free HBV DNA bioanalysis .
研究领域:
分子生物学
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医学诊断
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纳米技术
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DOI
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2023
·
IF 15.1
Yuqing Zhu,Xiaohe Zheng,Rui Zhu,Hongrui Zhao,Hongli Zhai,Feiyang Qian,Tenghua Zhang,Zihui Xie,Silan Liu,Bowen Jiang,Yan Sheng,Jiaming Hu
Detection of multiple biomarkers for accurate diagnosis of disease is highly desirable but remains challenging. Here, we developed a versatile diagnostic biosensor (SMARTLOCK) through the unique combination of DNA tetrahedron, nonlinear HCR and CRISPR-Cas12a system for simultaneous detection of multiple nucleic acids, small molecules, proteins and different types of combinations. Two DNA tetrahedrons are assembled into a functional dumbbell lock, which was unlocked by the target molecules, thereby triggering a two-stage signal amplification of nonlinear HCR and CRISPR-Cas12a to generate powerful fluorescence signals. The capability of multi-target simultaneous detection of SMARTLOCK is demonstrated in the detection of miR-141, miR-375, miR-21, ATP, cortisol, insulin and their combinations. SMARTLOCK could selectively (specificity = 93.3%) and sensitively (sensitivity = 90%) distinguish early-stage prostate cancer (PCa) patients (n = 10) from healthy donors (n = 30). Measurement of two PCa-related RNAs (miR-141 and miR-375) shows the potential of SMARTLOCK to be a highly accurate and low-cost tool for the screening, diagnosis and prognosis of various diseases.
研究领域:
生物技术
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医学诊断
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分子生物学
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产品关联
DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2024
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IF 10.7
Chuanyu Yang,Caiyi Du,Feiyu Yuan,Peitong Yu,Boxu Wang,Changshun Su,Ruiqi Zou,Junyang Wang,Xu Yan,Chunyan Sun,Hongxia Li
Benefiting from specific target recognition and trans-cleavage capabilities, the CRISPR/Cas12a system has great application prospects in the design of highly sensitive and rapid fluorescence biosensors. The CRISPR/Cas12a-based fluorophore-quencher molecular beacons exhibit single-color emission and are easily exposed to interference from environmental factors. Herein, we design a CRISPR/Cas12a-derived ratiometric fluorescence sensor for Pb 2+ detection based on embedded carbon dots@zeolitic imidazolate framework-8 (CDs@ZIF-8) composites and DNAzyme. The functions of ZIF-8 about encapsulating red emissive CDs in the inner cavity and adsorbing DNA on the outer surface are integrated to establish dual fluorescence signals, thereby reducing the possibility of interference and improving sensing accuracy. The presence of Pb 2+ is converted into the change of activator by the GR5 DNAzyme to activate the CRISPR/Cas12a system, which provides signal amplification through multiple turnovers of side branch cutting, achieving highly sensitive detection of Pb 2+ with a low detection limit of 18 pM. This method has the advantages of simplicity, universality, and excellent quantitative ability, and has broad prospects in sensing applications.
研究领域:
生物传感
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分子生物学
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环境科学
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DOI
PubMed
高影响因子
Nature Protocols
·
2014
·Vol. 9
·期 10
·2493-2512
·
IF 7.782
Shao Yanjiao,Guan Yuting,Wang Liren,Qiu Zhongwei,Liu Meizhen,Chen Yuting,Wu Lijuan,Li Yongmei,Ma Xueyun,Liu Mingyao,Li Dali
Conventional embryonic stem cell (ESC)–based gene targeting, zinc-finger nuclease (ZFN) and transcription activator–like effector nuclease (TALEN) technologies are powerful strategies for the generation of genetically modified animals. Recently, the CRISPR/Cas system has emerged as an efficient and convenient alternative to these approaches. We have used the CRISPR/Cas system to generate rat strains that carry mutations in multiple genes through direct injection of RNAs into one-cell embryos, demonstrating the high efficiency of Cas9-mediated gene editing in rats for simultaneous generation of compound gene mutant models. Here we describe a stepwise procedure for the generation of knockout and knock-in rats. This protocol provides guidelines for the selection of genomic targets, synthesis of guide RNAs, design and construction of homologous recombination (HR) template vectors, embryo microinjection, and detection of mutations and insertions in founders or their progeny. The procedure from target design to identification of founders can take as little as 6 weeks, of which <10 d is actual hands-on working time.
研究领域:
分子生物学
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遗传学
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实验动物学
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产品关联
DOI
PubMed
高影响因子
ACS Nano
·
2023
·
IF 17.1
Dou Wang,Xuedong Wang,Feidi Ye,Jin Zou,Jiuxin Qu,Xingyu Jiang
Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a “sample-in-answer-out” single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.
研究领域:
生物技术
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分子生物学
·
诊断方法
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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）