Biosensors-Basel
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2025
·Vol. 15
·期 12
·821
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IF 5.6
Jie Chen,Shutao Liu,Sunyi Chen,Jingwen Mai,Maiwula Abudukadi,Yao Chen,Jie Lu,Guanglei Li,Chenchen Ge
Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor’s response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application.
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
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2024
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IF 5.7
Tianyi Zhang,Xiaoqin Wang,Yingtao Jiang,Yunyun Zhang,Shuhao Zhao,Jian Hu,Fei Hu,Niancai Peng
Background Nucleic acid testing is the most effective detection method currently available for the diagnosis of respiratory infectious diseases. However, the conventional real-time fluorescent quantitative PCR technique, which is regarded as the gold standard method for nucleic acid detection, presents significant challenges for implementation in home self-testing and popularization in underdeveloped regions due to its rigorous experimental standards. It is therefore clear that an easy-to-use, miniaturized nucleic acid testing technology and products for nonprofessionals are of great necessity to define the pathogens and assist in controlling disease transmission. (87) Results In this study, we propose a strategy for self-testing of respiratory pathogen nucleic acid that is oriented towards the public and user-friendly. The proposed system integrates the processes of extraction-free nucleic acid release, RPA isothermal amplification, and CRISPR fluorescence detection into a compact configuration. A microfluidic testing chip actuated by air pouches and a battery/USB-powered reusable device has been developed to enable simultaneous detection of internal reference genes and viral targets in a fully enclosed condition. The system allows for sample-in, and result-out testing in less than 30 minutes with a detection limit of 2 copies/μL. Additionally, a straightforward signal-light-based result display method has been developed to make it easy and intuitive for users to access the results. Furthermore, freeze-drying reagent is introduced to guarantee the storage and transportation of testing chips in ambient conditions. (135) Significance This work presents a miniaturized, portable, and highly sensitive nucleic acid detection system, where simple operating procedures have been designed for unskilled users. It is our belief that the testing system developed in this work is well suited for home-based self-testing and infection diagnosis in resource-limited areas, due to the above-mentioned advantages. (52)
研究领域:
分子生物学
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医学检验
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生物工程
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PubMed
高影响因子
FOOD CONTROL
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2025
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IF 6.3
Yixiang Sun,Liren Zhang,Huimin Wu,Hongjun Chen,Huijie Hu,Chongyu Zhang,Xiaoqiang Li,Yuan Li,Yimai Wang,Liqiang Luo,Yizhi Song
Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a significant pathogen of global concern, presenting substantial public health risks through foodborne transmission and contributing to elevated morbidity and mortality rates worldwide. The critical need for precise and timely identification of MRSA in food matrices has become increasingly paramount for effective public health protection. While numerous CRISPR-based detection platforms for MRSA have been recently developed, their widespread implementation has been hindered by intricate multi-step protocols and dependency on sophisticated instrumentation. In this study, we developed a direct, accurate and visual detection method for MRSA — the RPA-Cas12a-LFS method. This method comprises two main components: (1) the direct one-pot RPA-Cas12a system, which integrates bacterial lysis, RPA nucleic acid amplification, and CRISPR-Cas12a nucleic acid detection into a single step performed simultaneously at a constant temperature, and (2) the streptavidin-gold nanoparticles (SA-AuNP)-based CRISPR-specific lateral flow strip (LFS). By eliminating the need for nucleic acid extraction, this method significantly simplifies the experimental procedure and reduces the risk of cross-contamination. Through systematic optimization, this method demonstrated exceptional performance, enabling direct and specific identification of MRSA at remarkably low concentrations (10 CFU/mL) within 60 min in various milk samples. This advanced detection method, characterized by its direct sample processing, exceptional accuracy, visual interpretability, cost-efficiency, and minimal equipment requirements, is particularly suitable for on-site and real-time monitoring of pathogenic bacteria in the food industry.
研究领域:
微生物学
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食品安全
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分子诊断
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DOI
高影响因子
LWT-FOOD SCIENCE AND TECHNOLOGY
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2025
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IF 6.6
Haibin Liu,Yi Yuan,Qiuyue Wang,Hang Tian,Yingxue Wu,Haijie Li,Hongshuo Chen,Lei Wang,Bingrui Liu
Meat adulteration has become a significant concern due to its potential to diminish the nutritional value and trigger allergic reactions. To address the issue of fraudulent adulteration in beef ( Bos taurus ) products, a visual strategy is proposed for multiplex detection of chicken ( Gallus gallus ), duck ( Anas platyrhynchos ), and pork ( Sus scrofa ) components in beef. First, the DNA could be easily extracted from the meat samples using the microneedle within 1 min and used as recombinase polymerase amplification (RPA) template. Next, three primer pairs specific to chicken, duck, and pork DNA are designed with unique oligonucleotide tags and a spacerC9 to halt polymerase activity, enabling the RPA amplicons to possess a single-stranded DNA tail. These tail sequences precisely hybridize with nanoparticle-labeled detection probes and pre-immobilized capture probes on test lines, generating a colorimetric signal on the lateral flow strip (LFS). The enhanced LFS utilizes three distinct Au/Ag nanoparticles, including Au@Ag (yellow), gold nanoflowers (cyan), and gold nanospheres (magenta), each targeting chicken, duck, and pork DNA, respectively. The test output can be easily interpreted through a “magenta yellow cyan” color code. The sensitivity of the multicolor LFS is further improved by the incorporation of DNA-Au/Ag nanoaggregates. Four functional oligonucleotides with complementary sequences are combined to form DNA-Au/Ag nano aggregates, allowing for increased coupling of Au/Ag and enhancing detection sensitivity. Cross-reactivity tests with ten non-target species demonstrate the excellent specificity of the assay. The method can detect chicken, duck, or pork at levels as low as 0.1 g/kg in simulated adulterated beef samples. The entire process, including rapid DNA extraction (1 min), RPA reaction (15 min), and LFS detection (5 min), is completed within 21 min. This platform is user-friendly, eliminating extensive training and specialized equipment, making it ideal for rapid on-site detection of multiplex adulteration by personnel in various fields.
研究领域:
生物传感器
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纳米技术
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食品安全与真伪鉴定
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分子诊断
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DNA杂交
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现场快速检测
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DOI
高影响因子
METHODS
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2021
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IF 3.608
Menglu Hu,Debin Zhu,Xiaoming Zhou
The construction of a rapid, simple, and specific nucleic acid detection platform is of great significance to the control of the large-scale spread of infectious diseases. We have recently established a magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system (termed M−CDC), which effectively integrates the advantages of CRISPR/Cas12a, magnetic beads-based separation, and AuNP bioprobe to provide a simple and specific biosensing platform for nucleic acid assay. The M−CDC method is compatible with point-of-care testing and enables the detection of nucleic acid samples in less than an hour without relying on expensive and complex instruments. In this paper, step-by-step instructions for M−CDC assay, including recombinase polymerase amplification (RPA)/reverse transcription-polymerase chain reaction (RT-RPA) of DNA or RNA, Cas12a-mediated target recognition and cleavage, and subsequent magnetic beads-mediated colorimetric readouts are provided. In addition, the protocol for the expression and purification of Lachnospiraceae bacterium -Cas12a (LbCas12a) protein, the design and synthesis of high-efficient crRNA, and the preparation of AuNP bioprobe are also offered.
研究领域:
生物技术
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分子生物学
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诊断方法
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纳米技术
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DOI
PubMed
高影响因子
Food Quality and Safety
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2025
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IF 4.4
Hu Jinyan,Liu Xiaogang,Liu Tiantian,Zhao Ting,Li Mingliang,Yang Qilin,Jiang Xiaofeng,Xiong Yanfei,Shen Caihong,Liu Shuangping,Mao Jian,Tu Rongkun
ObjectivesYeast plays a crucial role in fermented foods, yet the application of the CRISPR/Cas system for yeast detection has been rarely reported. The objective of this study is to develop a rapid and quantitative detection method for brewing yeast based on the CRISPR-Cas12a system, aiming to achieve higher sensitivity than the quantitative real-time polymerase chain reaction (qPCR) assay and fill the gap in the application of CRISPR for yeast detection in fermented foods.Materials and Methods: This work established one-step RPA-CRISPR/Cas12a (ORCC) detection for quantifying yeasts in fermented foods by integrating target amplification and the CRISPR system into a one-pot reaction.ResultsA numerical standardized definition was established for the quantifiable state in ORCC detection of yeasts. Using ORCC detection, which occurs at 37 °C, absolute quantification of Saccharomyces cerevisiae, Wickerhamomyces anomalus, and Zygosaccharomyces bailii can be completed in less than 45 min. Under optimal conditions, the limits of quantification and the limits of detection for three yeasts were 100 copies/μL and 1 copy/μL, with no cross-reaction observed among non-target yeasts. In pure yeast samples, ORCC detection of the target yeasts achieved a detection rate of 91.60% - 116.57%. In simulated fermentation samples, the recovery rate for spiked target yeasts using ORCC detection ranged from 92.78% to 106.90%. In original naturally fermented samples, target yeasts can be quantified by ORCC detection but not by qPCR assay. In spiked naturally fermented samples, the recovery rates of the qPCR assay were generally lower than those of ORCC detection.ConclusionORCC detection is simple, rapid, accurate, and highly sensitive for detecting three yeasts in fermented foods. It has significant potential for precisely monitoring yeast growth and fermentation process in various fermentation systems.
研究领域:
微生物学
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分子生物学
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食品科学
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DOI
高影响因子
ANALYTICAL CHEMISTRY
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2025
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IF 6.7
Fei Hu,Kaihui Liu,Yunyun Zhang,Shuhao Zhao,Tianyi Zhang,Cuiping Yao,Xing Lv,Jing Wang,Xiaolong Liu,Niancai Peng
Infectious diseases, especially respiratory infections, have been significant threats to human health. Therefore, it is essential to develop rapid, portable, and highly sensitive diagnostic methods for their control. Herein, a short-time preamplified, one-pot clustered regularly interspaced short palindromic repeats (CRISPR) nucleic acid detection method (SPOC) is developed by combining the rapid recombinase polymerase amplification (RPA) with CRISPR-Cas12a to reduce the mutual interference and achieve facile and rapid molecular diagnosis. SPOC can reduce the detection time and stably detect up to 1 copy/μL of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA without affecting the detection sensitivity. A highly sensitive one-pot assay integrated with reverse transcription RPA is achieved by wrapping paraffin with a specific melting point on the lyophilized CRISPR reagent surface. A self-heating pack is designed based on thermodynamic principles to melt the paraffin and release CRISPR reagents, enabling low-cost and time-saving detection. Notably, the designed system, coupled with RNA extraction-free technology, can achieve “sample-in-answer-out” detection of the SARS-CoV-2 Orf1ab gene within 22 min using smartphone imaging. The developed assay is validated on 12 clinical samples, and the results 100% correlate with real-time polymerase chain reaction. SPOC is time-saving, is easy to operate, and can eliminate centrifugal and complex hardware devices, satisfying the demand for point-of-care diagnostics in resource-constrained settings.
研究领域:
分子诊断学
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传染病学
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生物技术
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2021
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IF 6.986
Wei S. Zhang,Jianbin Pan,Feng Li,Min Zhu,Mengting Xu,Hongyan Zhu,Yanyan Yu,Gaoxing Su
The outbreak of the pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need for developing a facial and cost-effective detection method. The requirement of well-trained personnel and sophisticated instrument of current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder the practical application worldwide. In this regard, a reverse transcription recombinase polymerase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed for the SARS-CoV-2 detection. The methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as a universal colorimetric readout and can specifically target ORF1ab and N regions of the SARS-CoV-2 genome. After the virus genome is amplified through RT-RPA, the resulting abundant dsDNA will bind and activate Cas12a. Under trans-cleavage degradation, the capped DNA substrate will be hydrolyzed gradually from AuNPs, demonstrating a change in the surface plasmon resonance (SPR), which can be facially monitored by UV–vis absorbance spectroscopy and naked eye observation. The high amplification efficiency from RT-RPA and Cas12a trans-cleavage process bring the sensitivity of our method to 1 copy of viral genome sequence per test. Notably, under the dual variations inspecting from the isothermal amplification and Cas12a activation process, the false positive events from other beta coronavirus members can be effectively avoided and thus significantly improve the specificity. Furthermore, the reliability of this colorimetric assay is validated by standard clinical samples from the hospital laboratory department. Through integration of the inherently high sensitivity and specificity from an RPA-coupled Cas12a system with the intrinsic simplicity of AuNP-based colorimetric assay, our method increases the practical testing availability of SARS-CoV-2.
研究领域:
分子生物学
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病毒学
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诊断学
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DOI
PubMed
高影响因子
TALANTA
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2025
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IF 6.1
Mengqiu Xiong,Yao Wang,Shuhan Lu,Nasifu Lubanga,Tao Li,Zhihao Li,Bangshun He,Ying Li
Multiple infections of respiratory viruses are common in patients with clinical respiratory diseases, but current detection methods still have problems such as complex equipment and long detection time. Rapid, low-cost, and on-site detection of human respiratory viruses is crucial for both clinical diagnosis and population screening. In this research, we created a space-coded microfluidic chip (SC-Chip) for the recognition of nine respiratory viruses: influenza A virus, influenza B virus, severe acute respiratory syndrome coronavirus 2, human coronavirus OC43, human coronavirus NL63, human coronavirus HKU1, human respiratory syncytial virus, human parainfluenza virus, and human metapneumovirus. For the first time, a comprehensive sequence comparison among these viruses was performed to design the recombinase polymerase amplification (RPA) primers and Cas12a-crRNAs. The SC-Chip partitions samples amplified by RPA into spatially coded wells preloaded with CRISPR-Cas12a detection reagents, enabling the identification of all nine viral targets in a single test using a single fluorescence probe. The chip-based assay displays 9 respiratory viruses in less than 40 min with a minimum detection limit at a concentration of 10 −18 M (∼1 copy/reaction). Additionally, the efficacy of the method was assessed through its application to 35 clinical patient samples identified as being at risk for respiratory virus infection, yielding a sensitivity of 90 % and a specificity of 100 %. In summary, this space-coded microfluidic CRISPR system offers several advantages, including ease of operation, cost-effectiveness, and rapid data acquisition, thereby holding great potential for multiplexed detection of nucleic acid targets in a clinical setting.
研究领域:
分子生物学
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医学诊断
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微流控技术
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
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2023
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IF 6.2
Ling Deng,Xiaoyi He,Ke Liu,Yuanli Li,Han Xia,Hang Qian,Xiaoxue Lu,Xuhu Mao,Yang Xiang
Burkholderia pseudomallei is the causative agent of melioidosis, a potentially life-threatening infectious disease, and poses public health risks in endemic areas. Due to the high mortality, intrinsic antibiotic resistance , and atypical manifestations, establishing a rapid, accurate, and sensitive identification of B. pseudomallei enables earlier diagnosis, proper treatments, and better outcomes of melioidosis. Herein, we present a O ne- P ot C RISPR-integrated assay for I nstant and V isual D etection (termed OPC-IVD) of B. pseudomallei . The integration of recombinase polymerase amplification and CRISPR-Cas12a recognition-activated trans -cleavage, achieved a true all-in-one single-tube reaction system, initiating the amplification and cleavage simultaneously, which realized a facile sample-to-answer assay. This approach could be performed with simplified DNA extraction and completed around 30 min by holding the reaction tube in the hand. The detection limit of our OPC-IVD was determined to be 2.19 copy/uL of plasmid DNA, 12.5 CFU/mL of B. pseudomallei , and 61.5 CFU/mL of bacteria in spiked blood samples, respectively. Furthermore, the introduction of internal amplification control effectively reduced the occurrence of false negatives, which was incorporated in the reaction system, and amplified simultaneously with the target and read by naked eyes. The assay exhibited 100% accuracy when evaluated in clinical isolates and samples. The streamlined workflow of our OPC-IVD of B. pseudomallei enables a field-deployable, instrument-free, and ultra-fast approach that can be utilized by non-expert personnel in the field of molecular diagnosis of melioidosis especially in under-resourced setting.
研究领域:
医学诊断
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分子生物学
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传染病学
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产品关联
DOI
PubMed
高影响因子
POULTRY SCIENCE
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2024
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IF 3.8
Sha-Sha Chen,Yong-Lei Yang,Hong-Yun Wang,Tian-Kui Guo,Riaz-M Azeem,Chun-Wei Shi,Gui-Lian Yang,Hai-Bin Huang,Yan-Long Jiang,Jian-Zhong Wang,Xin Cao,Nan Wang,Yan Zeng,Wen-Tao Yang,Chun-Feng Wang
ABSTRACT Avian influenza virus (AIV) subtype H9N2 has significantly threatened the poultry business in recent years by having become the predominant subtype in flocks of chickens, ducks, and pigeons. In addition, the public health aspects of H9N2 AIV pose a significant threat to humans. Early and rapid diagnosis of H9N2 AIV is therefore of great importance. In this study, a new method for the detection of H9N2 AIV based on fluorescence intensity was successfully established using CRISPR/Cas13a technology. The Cas13a protein was first expressed in a prokaryotic system and purified using nickel ion affinity chromatography, resulting in a high-purity Cas13a protein. The best RPA(Recombinase polymerase amplification) primer pairs and crRNA were designed and screened, successfully constructing the detection of H9N2 AIV based on CRISPR/Cas13a technology. Optimal concentration of Cas13a and crRNA was determined to optimise the constructed assay. The sensitivity of the optimised detection system is excellent, with a minimum detection limit of 10° copies/μL and didn't react with other avian susceptible viruses, with excellent specificity. The detection method provides the basis for the field detection of the H9N2 AIV.
研究领域:
分子生物学
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病毒学
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生物技术
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公共卫生
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL BIOCHEMISTRY
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2019
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IF 2.507
Guang-Cheng Luo,Ting-Ting Yi,Bing Jiang,Xiao-lan Guo,Guo-Yuan Zhang
Recombinase polymerase amplification (RPA) is a widespread isothermal amplification method and regarded as an excellent candidate to replace polymerase chain reaction. However, the specificity of RPA is not always satisfactory when the sample contains amounts of background DNA . Herein, we report a novel RPA method named betaine-assisted RPA (B-RPA) that uses inexpensive betaine to avoid nonspecific amplification effectively. Result show that nonspecific amplification is prone to occur in RPA if the primers have not been rigorously refined, especially in detecting samples with large amounts of background DNA. This problem has been addressed by adding betaine to the RPA reactions. Our data show that the addition of 0.8 M betaine can significantly increase specificity and efficiency simultaneously. This B-RPA method is also used to detect hepatitis B virus DNA in clinical plasma samples, thereby demonstrating the clinical practicability of B-RPA.
研究领域:
分子生物学
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临床诊断
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病毒学
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DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）