Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,c...Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.This paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.展开更多
With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a c...With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a convenient and visual technique with low equipment requirements and high sensitivity for the field detection of GM plants is still lacking.On the basis of the existing recombinase polymerase amplification(RPA)technique,we developed a multiplex RPA(multi-RPA)method that can simultaneously detect three transgenic elements,including the cauliflower mosaic virus 35S gene(CaMV35S)promoter,neomycin phosphotransferaseⅡgene(NptⅡ)and hygromycin B phosphotransferase gene(Hyg),thus improving the detection rate.Moreover,we coupled this multi-RPA technique with the CRISPR/Cas12a reporter system,which enabled the detection results to be clearly observed by naked eyes under ultraviolet(UV)light(254 nm;which could be achieved by a portable UV flashlight),therefore establishing a multi-RPA visual detection technique.Compared with the traditional test strip detection method,this multi-RPA-CRISPR/Cas12a technique has the higher specificity,higher sensitivity,wider application range and lower cost.Compared with other polymerase chain reaction(PCR)techniques,it also has the advantages of low equipment requirements and visualization,making it a potentially feasible method for the field detection of GM plants.展开更多
Amplification-free,highly sensitive,and specific nucleic acid detection is crucial for health monitoring and diagnosis.The type III CRISPR-Cas10 system,which provides viral immunity through CRISPRassociated protein ef...Amplification-free,highly sensitive,and specific nucleic acid detection is crucial for health monitoring and diagnosis.The type III CRISPR-Cas10 system,which provides viral immunity through CRISPRassociated protein effectors,enables a new amplification-free nucleic acid diagnostic tool.In this study,we develop a CRISPR-graphene field-effect transistors(GFETs)biosensor by combining the type III CRISPR-Cas10 system with GFETs for direct nucleic acid detection.This biosensor exploits the target RNA-activated continuous ss DNA cleavage activity of the d Csm3 CRISPR-Cas10 effector and the high charge density of a hairpin DNA reporter on the GFET channel to achieve label-free,amplification-free,highly sensitive,and specific RNA detection.The CRISPR-GFET biosensor exhibits excellent performance in detecting medium-length RNAs and miRNAs,with detection limits at the aM level and a broad linear range of 10^(-15)to 10^(-11)M for RNAs and 10^(-15)to 10^(-9)M for miRNAs.It shows high sensitivity in throat swabs and serum samples,distinguishing between healthy individuals(N=5)and breast cancer patients(N=6)without the need for extraction,purification,or amplification.This platform mitigates risks associated with nucleic acid amplification and cross-contamination,making it a versatile and scalable diagnostic tool for molecular diagnostics in human health.展开更多
Glial fibrillary acidic protein(GFAP)is one of the discriminative biomarkers for diagnosing traumatic brain injury(TBI),and accurate determination of GFAP is clinically significant.In this study,a novel fluorescence i...Glial fibrillary acidic protein(GFAP)is one of the discriminative biomarkers for diagnosing traumatic brain injury(TBI),and accurate determination of GFAP is clinically significant.In this study,a novel fluorescence immunoassay system was designed.We encapsulated carbon dots with a high fluorescence quantum yield(QY=92.5%)inside silicon nanocapsules to serve as fluorescent markers.These markers were then integrated with the streptavidin(SA)-biotin biomagnification system and immunomagnetic separation technology for the sensitive detection of GFAP.Based on the signal cascade amplification effect of the silicon nanocapsules and SA-biotin,the fluorescence signal of the SA-biotin-modified immunofluorescence nanocapsules increased 3.6-fold compared to the carbon dot-based immunoprobe.The fluorescence immunoassay system was constructed for GFAP using SA-biotin-modified immunocapsules as the sensing probe and immunomagnetic nanoparticles as the immunorecognition probe.The fluorescence immunoassay system can specifically and ultra-sensitively quantify GFAP in blood samples,with a detection range of 10 pg/mL–10 ng/mL and detection limits of 3.2 pg/mL(serum)and 3.6 pg/mL(plasma).Moreover,the fluorescence immunoassay system exhibited prominent recoveries of 99.4%–100.4%(phosphate buffered saline),96%–102.6%(serum),and 93.2%–110.2%(plasma),with favorable specificity and excellent stabilization.The novel fluorescence immunoassay system provides a new approach to the clinical analysis of GFAP and may serve as a potential tool for screening and diagnosing TBI.展开更多
Lateral flow immunoassay(LFIA),a rapid detection technique noted for simplicity and economy,has showcased indispensable applicability in diverse domains such as disease screening,food safety,and environmental monitori...Lateral flow immunoassay(LFIA),a rapid detection technique noted for simplicity and economy,has showcased indispensable applicability in diverse domains such as disease screening,food safety,and environmental monitoring.Nevertheless,challenges still exist in detecting ultra-low concentration analytes due to the inherent sensitivity limitations of LFIA.Recently,significant advances have been achieved by integrating enzyme activity probes and transforming LFIA into a highly sensitive tool for rapidly detecting trace analyte concentrations.Specifically,modifying natural enzymes or engineered nanozymes allows them to function as immune probes,directly catalyzing the production of signal molecules or indirectly initiating enzyme activity.Therefore,the signal intensity and detection sensitivity of LFIA are markedly elevated.The present review undertakes a comprehensive examination of pertinent research literature,offering a systematic analysis of recently proposed enzyme-based signal amplification strategies.By way of comparative assessment,the merits and demerits of current approaches are delineated,along with the identification of research avenues that still need to be explored.It is anticipated that this critical overview will garner considerable attention within the biomedical and materials science communities,providing valuable direction and insight toward the advancement of high-performance LFIA technologies.展开更多
Development of accurate analytical protocols for cancer biomarkers is used for the initial prescreening of malignant tumors,disease surveillance,and efficacy assessment with significant clinical benefits.In this work,...Development of accurate analytical protocols for cancer biomarkers is used for the initial prescreening of malignant tumors,disease surveillance,and efficacy assessment with significant clinical benefits.In this work,we reported a liposome-mediated signal-off photoelectrochemical(PEC)immunoassay for the sensitive detection of carcinoembryonic antigen(CEA)using ternary transition metal sulfide CuS/ZnCdS as the photoactive material.Good photocurrents were acquired on the basis of specific oxidation reaction of dopamine on the CuS/ZnCdS.The energy band relationship of CuS/ZnCdS was determined,and the wellmatched oxidation potential of dopamine was verified.To achieve accurate recovery of low-abundance CEA,systematic PEC evaluation from human serum samples was performed by combining with classical immunoreaction and liposome-induced dopamine amplification strategy with high stability and selectivity.Under optimum conditions,PEC immunoassay displayed good photocurrent responses toward target CEA with a dynamic linear range of 0.1-50 ng/mL with a detection limit of 31.6 pg/mL.Importantly,this system by combining with a discussion of energy level matching between semiconductor energy bands and small-molecules opens a new horizon for development of high-efficient PEC immunoassays.展开更多
Surface irregularities,such as hills and ridges,can significantly amplify ground motion caused by earthquakes.Therefore,in this study,we propose an analytical solution model to investigate the interaction between an a...Surface irregularities,such as hills and ridges,can significantly amplify ground motion caused by earthquakes.Therefore,in this study,we propose an analytical solution model to investigate the interaction between an asymmetric triangular hill on Earth and SH waves.Firstly,based on the development of wave functions and regional matching techniques,we introduce a semi-circular artificial auxiliary boundary,dividing the solution model into a semi-infinite body containing a semi-circular depression and an asymmetric fan-shaped region.Secondly,we derive the domain function form applicable to solving asymmetric problems.Utilizing the theory of complex variables,we establish a well-posed matrix for solving domain functions within the same coordinate system.Numerical results demonstrate that the scattering of SH waves by a protuberance is jointly influenced by the geometric parameters of the hill and the angle of incidence.Additionally,the frequency of the incident wave also has a certain degree of impact on the displacement amplitude.This study elucidates the scattering mechanism of SH waves by complex boundaries,providing a theoretical reference for building site selection and seismic design.In practical problems,the asymmetric assumption is more applicable than the symmetry assumption.展开更多
Although diverse signal-amplified methods have been committed to improve the sensitivity of surface plasmon resonance(SPR)biosensing,introducing convenient and robust signal amplification strategy into SPR biosensing ...Although diverse signal-amplified methods have been committed to improve the sensitivity of surface plasmon resonance(SPR)biosensing,introducing convenient and robust signal amplification strategy into SPR biosensing remains challenging.Here,a novel nanozyme-triggered polymerization amplification strategy was proposed for constructing highly sensitive surface plasmon resonance(SPR)immunosensor.In detail,Au@Pd core-shell nanooctahedra nanozyme with superior peroxidase(POD)-like activity was synthesized and utilized as a label probe.Simultaneously,Au@Pd core-shell nanooctahedra nanozyme can catalyze the decomposition of H_(2)O_(2)to form hydroxyl radicals(·OH)that triggers the polymerization of aniline to form polyaniline attaching on the surface of sensor chip,significantly amplifying SPR responses.The sensitivity of SPR immunosensor was enhanced by nanozyme-triggered polymerization amplification strategy.Using human immunoglobulin G(HIgG)as a model,the constructed SPR immunosensor obtains a wide linear range of 0.005–1.0μg/m L with low detection limit of 0.106 ng/m L.This research provides new sights on establishing sensitive SPR immunosensor and may evokes more inspiration for developing signal amplification methods based on nanozyme in biosensing.展开更多
Chemodynamic therapy(CDT),using Fenton agents to generate highly cytotoxic•OH from H_(2)O_(2)has been demonstrated as a powerful anticancer method.However,the insufficient endogenous H_(2)O_(2)in tumor cells greatly l...Chemodynamic therapy(CDT),using Fenton agents to generate highly cytotoxic•OH from H_(2)O_(2)has been demonstrated as a powerful anticancer method.However,the insufficient endogenous H_(2)O_(2)in tumor cells greatly limited its therapeutic effect.Herein,we prepared a pH-responsiveβ-lapachone-loaded ironpolyphenol nanocomplex(LIPN)through a one-pot method.β-Lapachone in LIPN selectively enhanced H_(2)O_(2)concentration in tumor cells,and ferrous ions cascadely generated abundant cytotoxic•OH.Therefore,LIPN with cascade amplification of reactive oxygen species(ROS)showed high chemodynamic cytotoxicity in tumor cells,efficiently improving the expression of damage-associated molecular patterns(DAMPs),and exerting strong immunogenic cell death(ICD).As a result,LIPN exhibited efficient tumor inhibition ability in 4T1 subcutaneous tumor model in vivo with great biocompatibility.Additionally,the infiltration of cytotoxic CD8^(+)T lymphocytes and inhibition of regulatory CD4^(+)FoxP3^(+)T lymphocytes in tumors demonstrated the activation of immunosuppressive tumor microenvironment by LIPN-induced ICD.Therefore,this work provided a new approach to enhance ICD of chemodynamic therapy through selective cascade amplification of ROS in cancer cells.展开更多
Porcine Contagious Pleuropneumonia(PCP)is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae.The disease has been prevalent in pig farms since it was first identified in 1957(Pattison e...Porcine Contagious Pleuropneumonia(PCP)is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae.The disease has been prevalent in pig farms since it was first identified in 1957(Pattison et al.1957).展开更多
Bacterial blight(BB) is a devastating worldwide rice disease caused by Xanthomonas oryzae pv. oryzae(Xoo), which is difficult to diagnose based on early symptoms. Conventional chemical control yields limited effective...Bacterial blight(BB) is a devastating worldwide rice disease caused by Xanthomonas oryzae pv. oryzae(Xoo), which is difficult to diagnose based on early symptoms. Conventional chemical control yields limited effectiveness once BB has spread. Consequently, it is imperative to develop a rapid, highly sensitive, specific, and easy-to-use detection technique for early on-site diagnosis of BB. We first developed a recombinase-aided amplification-lateral flow dipstick(RAA-LFD) technique for the on-site detection of Xoo. The optimized reaction temperature and time were 37 ℃ and 20 min, indicating that the reaction system can be initiated by body temperature independently of any precision instruments. Evaluation of the RAA-LFD technique using the primers(RAAF2/R2) and probe(RAA2-nfo-probe) derived from the Xoo ORF0080 locus exhibited high specificity and eliminated cross-reactivity with other bacterial species. The sensitivity of RAA-LFD is up to 1 pg/μL for Xoo genomic DNA and 100 CFU/m L for Xoo cells. Significantly, this technique accurately detected Xoo from both artificially inoculated and naturally infected rice leaves at the early stage of infection, directly deploying plant tissue fluid as the template without DNA extraction. These attributes make the developed RAA-LFD system a viable technique for the early diagnosis of BB in the field, providing technical support for early-warning systems and disease control.展开更多
To capture the nonlinear dynamics and gain evolution in chirped pulse amplification(CPA)systems,the split-step Fourier method and the fourth-order Runge–Kutta method are integrated to iteratively address the generali...To capture the nonlinear dynamics and gain evolution in chirped pulse amplification(CPA)systems,the split-step Fourier method and the fourth-order Runge–Kutta method are integrated to iteratively address the generalized nonlinear Schrödinger equation and the rate equations.However,this approach is burdened by substantial computational demands,resulting in significant time expenditures.In the context of intelligent laser optimization and inverse design,the necessity for numerous simulations further exacerbates this issue,highlighting the need for fast and accurate simulation methodologies.Here,we introduce an end-to-end model augmented with active learning(E2E-AL)with decent generalization through different dedicated embedding methods over various parameters.On an identical computational platform,the artificial intelligence–driven model is 2000 times faster than the conventional simulation method.Benefiting from the active learning strategy,the E2E-AL model achieves decent precision with only two-thirds of the training samples compared with the case without such a strategy.Furthermore,we demonstrate a multi-objective inverse design of the CPA systems enabled by the E2E-AL model.The E2E-AL framework manifests the potential of becoming a standard approach for the rapid and accurate modeling of ultrafast lasers and is readily extended to simulate other complex systems.展开更多
DNA repair enzymes are important in the repair of DNA lesions for maintaining the genome stability,and their abnormal expression induced various human cancers.Simultaneous detection of these DNA enzymes could provide ...DNA repair enzymes are important in the repair of DNA lesions for maintaining the genome stability,and their abnormal expression induced various human cancers.Simultaneous detection of these DNA enzymes could provide convincing evidence based on the comparison of the activity of multiple enzymes than on that of single enzyme.Although fluorescence approach has been applied for the simultaneous detection both of DNA repair enzymes,the spectral overlap and multiwavelength excitation severely restrict the number of available fluorophores.Thus,it is difficult to simultaneously detect three enzymes in a single analysis by fluorescence detection.Herein,we developed a method for the simultaneous determination of three DNA repair enzymes including human flap DNA endonuclease 1(FEN1),human alkyladenine DNA glycosylase(hAAG)and uracil DNA glycosylase(UDG)based on the combination of template-free amplification system with capillary electrophoresis-laser induced fluorescence(CE-LIF)detection.The amplification system was adopted to transfer and amplify the enzymatic products into different length DNA fragments which could be separated effectively by CE-LIF without the complicated modification of the capillary inner wall or labeling different tails on signal probes for separation.The method demonstrated a detection limit of 0.07 U/mL(0.08-160 U/mL)for FEN1,2.40 U/mL(2.5-250U/mL)for hAAG and 2.1×10^(-4)U/mL(0.0004-2.5 U/mL)for UDG,the relative standard deviations(RSDs)of peak time and peak area for different analytes were as follows:2.50%-4,37%and 3.24%-7.18%(inter-day);1.37%-2.71%and 1.43%-3.02%(intra-day),4.28%-6.08%and 4.16%-7.57%(column to column),respectively.And it can identify the inhibitor-like drugs,evaluate enzymatic kinetics and achieve the detection of three enzymes in cell extracts,providing a simple and powerful platform for simultaneous detection of more DNA repair enzymes.展开更多
Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited dis...Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited disorders.This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s.Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD,but there are some inevitable shortcomings limiting the scope of genetic diagnosis.Fortunately,different whole genome amplification (WGA) techniques have been developed to overcome these problems.Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed.Moreover,WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis.In this review,we will focus on the currently available WGA techniques and their applications,as well as the new technical trends from WGA products.展开更多
[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 su...[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.展开更多
[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional tou...[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional touchdown PCR and the optimized abnormal touchdown PCR were respectively carried out to amplify the genomic DNA of Cyprinus carpio.[Result] Only one evident electrophoretic band and a few Sox genes were obtained by using normal PCR;no obvious electrophoretic band but dispersive product was obtained by normal touchdown PCR;ideal result was obtained by the abnormal touchdown PCR that three evident electrophoretic bands and much more Sox genes were amplified.[Conclusion] The research provided theoretical basis for the optimization and selection of PCR amplification conditions of the large-family genes.展开更多
Objective] This study aimed to develop a reverse transcription loop-medi-ated isothermal amplification （RT-LAMP） method for detecting BVDV. [Method] Since gp48 gene of BVDV is among the most conserved regions, a set...Objective] This study aimed to develop a reverse transcription loop-medi-ated isothermal amplification （RT-LAMP） method for detecting BVDV. [Method] Since gp48 gene of BVDV is among the most conserved regions, a set of four primers was designed to amplify six target sequences at the gp48 gene region for the RT-LAMP assay. The optimization of the RT-LAMP reaction was performed by evaluat-ing reaction temperature and reaction time. [Result] The RT-LAMP aasay was suc-cessful y conducted at 56 ℃ within 40 min under isothermal conditions, and the re-sults could be detected as ladder-like bands using agarose gel electrophoresis. The RT-LAMP assay is highly sensitive and able to detect 3.74 ×100 copies/μl of BVDV RNA, as no cross-reaction was observed with other viruses. [Conclusion] Overal , the newly established RT-LAMP assay indicates the potential application in both clinical diagnosis and field surveil ance of BVDV.展开更多
Objective To establish an ultra-sensitive,ultra-fast,visible detection method for Vibrio parahaemolyticus(VP).Methods We established a new method for detecting the tdh and trh genes of VP using clustered regularly int...Objective To establish an ultra-sensitive,ultra-fast,visible detection method for Vibrio parahaemolyticus(VP).Methods We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a(CRISPR/Cas12a)combined with recombinase polymerase amplification and visual detection(CRISPR/Cas12a-VD).Results CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10^(-18)M(single molecule detection)within 30 min without cross-reactivity against other bacteria.When detecting pure cultures of VP,the consistency of results reached 100%compared with real-time PCR.The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10^(2)CFU/g.Conclusion The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods,such as real-time PCR,and has great potential for preventing the spread of pathogens.展开更多
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of fo...Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications are summarized in this article.展开更多
Pseudomonas aeruginosa(PA)is an opportunistic pathogen of humans and animals and a common source of nosocomial infections especially of the respiratory tract.Pseudomonas aeruginosa is also a major bacterial disease of...Pseudomonas aeruginosa(PA)is an opportunistic pathogen of humans and animals and a common source of nosocomial infections especially of the respiratory tract.Pseudomonas aeruginosa is also a major bacterial disease of poultry and in particular,eggs and newly hatched chicks.In this study,we developed a simple,accurate and rapid molecular detection method using cross priming amplification(CPA)with a nucleic acid test strip to detect P.aeruginosa.The assay efficiently amplified the target gene within 45 min at 62℃only using a simple water bath.The detection limit of the method was 1.18x 102 copiesμL^-1 for plasmid DNA and 4.4 CFU mL^-1 for bacteria in pure culture,and was 100 times more sensitive than conventional PCR.We screened 83 clinical samples from yellow-feather broiler breeder chickens and hospitalized/treated dogs and cats using CPA,PCR and traditional culture methods.The positive sample ratios were 15.3%(13/83)by CPA,13.3%(11/83)by PCR and 12.1%(10/83)by the culture method.The established CPA method has significant advantages for detecting P.aeruginosa.The method is easy to use and possesses high specificity and sensitivity without the requirements of complicated experimental equipment.The PA-CPA assay is especially fit for outdoor and primary medical units and is an ideal system for the rapid detection and monitoring of P.aeruginosa.展开更多
基金supported by grants from Jiangsu Higher Education Institution Innovative Research Team for Science and Technology(2021),the Key Technology Program of Suzhou People’s Livelihood Technology Projects(Grant Nos.SKY2021029,SZS2020311)the Open Project of Jiangsu Biobank of Clinical Resources(TC2021B009)the Qing-Lan Project of Jiangsu Province in China(2021,2022).
文摘Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.This paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.
基金the Experimental Technology Research Project of Zhejiang University(SYB202138)National Natural Science Foundation of China(32000195)。
文摘With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a convenient and visual technique with low equipment requirements and high sensitivity for the field detection of GM plants is still lacking.On the basis of the existing recombinase polymerase amplification(RPA)technique,we developed a multiplex RPA(multi-RPA)method that can simultaneously detect three transgenic elements,including the cauliflower mosaic virus 35S gene(CaMV35S)promoter,neomycin phosphotransferaseⅡgene(NptⅡ)and hygromycin B phosphotransferase gene(Hyg),thus improving the detection rate.Moreover,we coupled this multi-RPA technique with the CRISPR/Cas12a reporter system,which enabled the detection results to be clearly observed by naked eyes under ultraviolet(UV)light(254 nm;which could be achieved by a portable UV flashlight),therefore establishing a multi-RPA visual detection technique.Compared with the traditional test strip detection method,this multi-RPA-CRISPR/Cas12a technique has the higher specificity,higher sensitivity,wider application range and lower cost.Compared with other polymerase chain reaction(PCR)techniques,it also has the advantages of low equipment requirements and visualization,making it a potentially feasible method for the field detection of GM plants.
基金financially supported by the National Science and Technology Innovation 2030 Grants(2021ZD0201600)the National Key R&D Program of China(2021YFA0717000)+2 种基金the Intramural Joint Program Fund of State Key Laboratory of Microbial Technology(Project No.SKLMTIJP-2024-05)the Natural Science Foundation of Qingdao-Original exploration project(Project No.24-4-4-zrjj-139-jch)the National Natural Science Foundation of China(31771380)。
文摘Amplification-free,highly sensitive,and specific nucleic acid detection is crucial for health monitoring and diagnosis.The type III CRISPR-Cas10 system,which provides viral immunity through CRISPRassociated protein effectors,enables a new amplification-free nucleic acid diagnostic tool.In this study,we develop a CRISPR-graphene field-effect transistors(GFETs)biosensor by combining the type III CRISPR-Cas10 system with GFETs for direct nucleic acid detection.This biosensor exploits the target RNA-activated continuous ss DNA cleavage activity of the d Csm3 CRISPR-Cas10 effector and the high charge density of a hairpin DNA reporter on the GFET channel to achieve label-free,amplification-free,highly sensitive,and specific RNA detection.The CRISPR-GFET biosensor exhibits excellent performance in detecting medium-length RNAs and miRNAs,with detection limits at the aM level and a broad linear range of 10^(-15)to 10^(-11)M for RNAs and 10^(-15)to 10^(-9)M for miRNAs.It shows high sensitivity in throat swabs and serum samples,distinguishing between healthy individuals(N=5)and breast cancer patients(N=6)without the need for extraction,purification,or amplification.This platform mitigates risks associated with nucleic acid amplification and cross-contamination,making it a versatile and scalable diagnostic tool for molecular diagnostics in human health.
基金supported by the AMS Funding Project(No.ZZB2023C7010).
文摘Glial fibrillary acidic protein(GFAP)is one of the discriminative biomarkers for diagnosing traumatic brain injury(TBI),and accurate determination of GFAP is clinically significant.In this study,a novel fluorescence immunoassay system was designed.We encapsulated carbon dots with a high fluorescence quantum yield(QY=92.5%)inside silicon nanocapsules to serve as fluorescent markers.These markers were then integrated with the streptavidin(SA)-biotin biomagnification system and immunomagnetic separation technology for the sensitive detection of GFAP.Based on the signal cascade amplification effect of the silicon nanocapsules and SA-biotin,the fluorescence signal of the SA-biotin-modified immunofluorescence nanocapsules increased 3.6-fold compared to the carbon dot-based immunoprobe.The fluorescence immunoassay system was constructed for GFAP using SA-biotin-modified immunocapsules as the sensing probe and immunomagnetic nanoparticles as the immunorecognition probe.The fluorescence immunoassay system can specifically and ultra-sensitively quantify GFAP in blood samples,with a detection range of 10 pg/mL–10 ng/mL and detection limits of 3.2 pg/mL(serum)and 3.6 pg/mL(plasma).Moreover,the fluorescence immunoassay system exhibited prominent recoveries of 99.4%–100.4%(phosphate buffered saline),96%–102.6%(serum),and 93.2%–110.2%(plasma),with favorable specificity and excellent stabilization.The novel fluorescence immunoassay system provides a new approach to the clinical analysis of GFAP and may serve as a potential tool for screening and diagnosing TBI.
基金Financial supports from the National Natural Science Foundation of China(NSFC,Nos.52272144 and 22205048)Heilongjiang Provincial Natural Science Foundation of China(No.JQ2022E001)+3 种基金China Postdoctoral Science Foundation(Nos.2022M710931 and 2023T160154)Heilongjiang Postdoctoral Science Foundation(No.LBH-Z22010)Natural Science Foundation of Shandong Province(No.ZR2020ZD42)the Fundamental Research funds for the Central Universities are greatly acknowledged.
文摘Lateral flow immunoassay(LFIA),a rapid detection technique noted for simplicity and economy,has showcased indispensable applicability in diverse domains such as disease screening,food safety,and environmental monitoring.Nevertheless,challenges still exist in detecting ultra-low concentration analytes due to the inherent sensitivity limitations of LFIA.Recently,significant advances have been achieved by integrating enzyme activity probes and transforming LFIA into a highly sensitive tool for rapidly detecting trace analyte concentrations.Specifically,modifying natural enzymes or engineered nanozymes allows them to function as immune probes,directly catalyzing the production of signal molecules or indirectly initiating enzyme activity.Therefore,the signal intensity and detection sensitivity of LFIA are markedly elevated.The present review undertakes a comprehensive examination of pertinent research literature,offering a systematic analysis of recently proposed enzyme-based signal amplification strategies.By way of comparative assessment,the merits and demerits of current approaches are delineated,along with the identification of research avenues that still need to be explored.It is anticipated that this critical overview will garner considerable attention within the biomedical and materials science communities,providing valuable direction and insight toward the advancement of high-performance LFIA technologies.
基金financial support from the National Natural Science Foundation of China(Nos.22274022 and 21874022).
文摘Development of accurate analytical protocols for cancer biomarkers is used for the initial prescreening of malignant tumors,disease surveillance,and efficacy assessment with significant clinical benefits.In this work,we reported a liposome-mediated signal-off photoelectrochemical(PEC)immunoassay for the sensitive detection of carcinoembryonic antigen(CEA)using ternary transition metal sulfide CuS/ZnCdS as the photoactive material.Good photocurrents were acquired on the basis of specific oxidation reaction of dopamine on the CuS/ZnCdS.The energy band relationship of CuS/ZnCdS was determined,and the wellmatched oxidation potential of dopamine was verified.To achieve accurate recovery of low-abundance CEA,systematic PEC evaluation from human serum samples was performed by combining with classical immunoreaction and liposome-induced dopamine amplification strategy with high stability and selectivity.Under optimum conditions,PEC immunoassay displayed good photocurrent responses toward target CEA with a dynamic linear range of 0.1-50 ng/mL with a detection limit of 31.6 pg/mL.Importantly,this system by combining with a discussion of energy level matching between semiconductor energy bands and small-molecules opens a new horizon for development of high-efficient PEC immunoassays.
基金supported by the National Key R&D Program of China(Grant No.2022YFC3003601)Joint Funds of the National Natural Science Foundation of China Project on Earthquake Science(Grant No.U2239252)the program of the Innovative Research Team in China Earthquake Administration.
文摘Surface irregularities,such as hills and ridges,can significantly amplify ground motion caused by earthquakes.Therefore,in this study,we propose an analytical solution model to investigate the interaction between an asymmetric triangular hill on Earth and SH waves.Firstly,based on the development of wave functions and regional matching techniques,we introduce a semi-circular artificial auxiliary boundary,dividing the solution model into a semi-infinite body containing a semi-circular depression and an asymmetric fan-shaped region.Secondly,we derive the domain function form applicable to solving asymmetric problems.Utilizing the theory of complex variables,we establish a well-posed matrix for solving domain functions within the same coordinate system.Numerical results demonstrate that the scattering of SH waves by a protuberance is jointly influenced by the geometric parameters of the hill and the angle of incidence.Additionally,the frequency of the incident wave also has a certain degree of impact on the displacement amplitude.This study elucidates the scattering mechanism of SH waves by complex boundaries,providing a theoretical reference for building site selection and seismic design.In practical problems,the asymmetric assumption is more applicable than the symmetry assumption.
基金supported by National Natural Science Foundation of China(Nos.22474124,21575125)the National Natural Science Foundation of Jiangsu Province(No.BK20221370)+4 种基金Key University Natural Science Foundation of Jiangsu-Province(No.20KJA150004)the Project for Science and Technology of Yangzhou(No.YZ2022074)Project for Yangzhou City and Yangzhou University corporation(No.YZ2023204)the Open Research Fund of State Key Laboratory of Analytical Chemistry for Life Science(No.SKLACLS2405)Postgraduate Research&Practice Innovation Program of Jiangsu Province(No.KYCX22_3462)。
文摘Although diverse signal-amplified methods have been committed to improve the sensitivity of surface plasmon resonance(SPR)biosensing,introducing convenient and robust signal amplification strategy into SPR biosensing remains challenging.Here,a novel nanozyme-triggered polymerization amplification strategy was proposed for constructing highly sensitive surface plasmon resonance(SPR)immunosensor.In detail,Au@Pd core-shell nanooctahedra nanozyme with superior peroxidase(POD)-like activity was synthesized and utilized as a label probe.Simultaneously,Au@Pd core-shell nanooctahedra nanozyme can catalyze the decomposition of H_(2)O_(2)to form hydroxyl radicals(·OH)that triggers the polymerization of aniline to form polyaniline attaching on the surface of sensor chip,significantly amplifying SPR responses.The sensitivity of SPR immunosensor was enhanced by nanozyme-triggered polymerization amplification strategy.Using human immunoglobulin G(HIgG)as a model,the constructed SPR immunosensor obtains a wide linear range of 0.005–1.0μg/m L with low detection limit of 0.106 ng/m L.This research provides new sights on establishing sensitive SPR immunosensor and may evokes more inspiration for developing signal amplification methods based on nanozyme in biosensing.
基金supported by the National Natural Science Foundation of China(Nos.T2293753,52203194)the National Key R&D Program of China(No.2021YFA1201200)+1 种基金the Natural Science Foundation of Zhejiang Province(No.LR18E030002)2023 Hangzhou West Lake Pearl Project Leading Innovative Youth Team Project.
文摘Chemodynamic therapy(CDT),using Fenton agents to generate highly cytotoxic•OH from H_(2)O_(2)has been demonstrated as a powerful anticancer method.However,the insufficient endogenous H_(2)O_(2)in tumor cells greatly limited its therapeutic effect.Herein,we prepared a pH-responsiveβ-lapachone-loaded ironpolyphenol nanocomplex(LIPN)through a one-pot method.β-Lapachone in LIPN selectively enhanced H_(2)O_(2)concentration in tumor cells,and ferrous ions cascadely generated abundant cytotoxic•OH.Therefore,LIPN with cascade amplification of reactive oxygen species(ROS)showed high chemodynamic cytotoxicity in tumor cells,efficiently improving the expression of damage-associated molecular patterns(DAMPs),and exerting strong immunogenic cell death(ICD).As a result,LIPN exhibited efficient tumor inhibition ability in 4T1 subcutaneous tumor model in vivo with great biocompatibility.Additionally,the infiltration of cytotoxic CD8^(+)T lymphocytes and inhibition of regulatory CD4^(+)FoxP3^(+)T lymphocytes in tumors demonstrated the activation of immunosuppressive tumor microenvironment by LIPN-induced ICD.Therefore,this work provided a new approach to enhance ICD of chemodynamic therapy through selective cascade amplification of ROS in cancer cells.
基金supported by the University-Industry Col aborative Education Program,China(220904860093831)。
文摘Porcine Contagious Pleuropneumonia(PCP)is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae.The disease has been prevalent in pig farms since it was first identified in 1957(Pattison et al.1957).
基金supported by the Zhejiang Provincial Natural Science Foundation of China(Grant Nos.LY23C130004 and LZ24C130004)the National Natural Science Foundation of China(Grant No.32472115)+1 种基金the National Key Research and Development Program of China(Grant No.2022YFF1003301)the Agricultural Sciences and Technologies Innovation Program of Chinese Academy of Agricultural Sciences。
文摘Bacterial blight(BB) is a devastating worldwide rice disease caused by Xanthomonas oryzae pv. oryzae(Xoo), which is difficult to diagnose based on early symptoms. Conventional chemical control yields limited effectiveness once BB has spread. Consequently, it is imperative to develop a rapid, highly sensitive, specific, and easy-to-use detection technique for early on-site diagnosis of BB. We first developed a recombinase-aided amplification-lateral flow dipstick(RAA-LFD) technique for the on-site detection of Xoo. The optimized reaction temperature and time were 37 ℃ and 20 min, indicating that the reaction system can be initiated by body temperature independently of any precision instruments. Evaluation of the RAA-LFD technique using the primers(RAAF2/R2) and probe(RAA2-nfo-probe) derived from the Xoo ORF0080 locus exhibited high specificity and eliminated cross-reactivity with other bacterial species. The sensitivity of RAA-LFD is up to 1 pg/μL for Xoo genomic DNA and 100 CFU/m L for Xoo cells. Significantly, this technique accurately detected Xoo from both artificially inoculated and naturally infected rice leaves at the early stage of infection, directly deploying plant tissue fluid as the template without DNA extraction. These attributes make the developed RAA-LFD system a viable technique for the early diagnosis of BB in the field, providing technical support for early-warning systems and disease control.
基金supported by the National Natural Science Foundation of China(Grant Nos.62227821,62025503,and 62205199).
文摘To capture the nonlinear dynamics and gain evolution in chirped pulse amplification(CPA)systems,the split-step Fourier method and the fourth-order Runge–Kutta method are integrated to iteratively address the generalized nonlinear Schrödinger equation and the rate equations.However,this approach is burdened by substantial computational demands,resulting in significant time expenditures.In the context of intelligent laser optimization and inverse design,the necessity for numerous simulations further exacerbates this issue,highlighting the need for fast and accurate simulation methodologies.Here,we introduce an end-to-end model augmented with active learning(E2E-AL)with decent generalization through different dedicated embedding methods over various parameters.On an identical computational platform,the artificial intelligence–driven model is 2000 times faster than the conventional simulation method.Benefiting from the active learning strategy,the E2E-AL model achieves decent precision with only two-thirds of the training samples compared with the case without such a strategy.Furthermore,we demonstrate a multi-objective inverse design of the CPA systems enabled by the E2E-AL model.The E2E-AL framework manifests the potential of becoming a standard approach for the rapid and accurate modeling of ultrafast lasers and is readily extended to simulate other complex systems.
基金supported by the National Natural Science Foundation of China(Nos.21874060 and 22174058,U21A20282)the Science and Technology program of Gansu Province(No.22JR5RA476)。
文摘DNA repair enzymes are important in the repair of DNA lesions for maintaining the genome stability,and their abnormal expression induced various human cancers.Simultaneous detection of these DNA enzymes could provide convincing evidence based on the comparison of the activity of multiple enzymes than on that of single enzyme.Although fluorescence approach has been applied for the simultaneous detection both of DNA repair enzymes,the spectral overlap and multiwavelength excitation severely restrict the number of available fluorophores.Thus,it is difficult to simultaneously detect three enzymes in a single analysis by fluorescence detection.Herein,we developed a method for the simultaneous determination of three DNA repair enzymes including human flap DNA endonuclease 1(FEN1),human alkyladenine DNA glycosylase(hAAG)and uracil DNA glycosylase(UDG)based on the combination of template-free amplification system with capillary electrophoresis-laser induced fluorescence(CE-LIF)detection.The amplification system was adopted to transfer and amplify the enzymatic products into different length DNA fragments which could be separated effectively by CE-LIF without the complicated modification of the capillary inner wall or labeling different tails on signal probes for separation.The method demonstrated a detection limit of 0.07 U/mL(0.08-160 U/mL)for FEN1,2.40 U/mL(2.5-250U/mL)for hAAG and 2.1×10^(-4)U/mL(0.0004-2.5 U/mL)for UDG,the relative standard deviations(RSDs)of peak time and peak area for different analytes were as follows:2.50%-4,37%and 3.24%-7.18%(inter-day);1.37%-2.71%and 1.43%-3.02%(intra-day),4.28%-6.08%and 4.16%-7.57%(column to column),respectively.And it can identify the inhibitor-like drugs,evaluate enzymatic kinetics and achieve the detection of three enzymes in cell extracts,providing a simple and powerful platform for simultaneous detection of more DNA repair enzymes.
基金Project supported by the National Basic Research Program (973) of China (No.2007CB948104)the Natural Science Foundation of Zhejiang Province,China (No.Z207021)
文摘Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited disorders.This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s.Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD,but there are some inevitable shortcomings limiting the scope of genetic diagnosis.Fortunately,different whole genome amplification (WGA) techniques have been developed to overcome these problems.Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed.Moreover,WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis.In this review,we will focus on the currently available WGA techniques and their applications,as well as the new technical trends from WGA products.
基金Supported by Important Project of Jinlin Provincial Science and Technology Department(20065020)~~
文摘[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.
文摘[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional touchdown PCR and the optimized abnormal touchdown PCR were respectively carried out to amplify the genomic DNA of Cyprinus carpio.[Result] Only one evident electrophoretic band and a few Sox genes were obtained by using normal PCR;no obvious electrophoretic band but dispersive product was obtained by normal touchdown PCR;ideal result was obtained by the abnormal touchdown PCR that three evident electrophoretic bands and much more Sox genes were amplified.[Conclusion] The research provided theoretical basis for the optimization and selection of PCR amplification conditions of the large-family genes.
文摘Objective] This study aimed to develop a reverse transcription loop-medi-ated isothermal amplification （RT-LAMP） method for detecting BVDV. [Method] Since gp48 gene of BVDV is among the most conserved regions, a set of four primers was designed to amplify six target sequences at the gp48 gene region for the RT-LAMP assay. The optimization of the RT-LAMP reaction was performed by evaluat-ing reaction temperature and reaction time. [Result] The RT-LAMP aasay was suc-cessful y conducted at 56 ℃ within 40 min under isothermal conditions, and the re-sults could be detected as ladder-like bands using agarose gel electrophoresis. The RT-LAMP assay is highly sensitive and able to detect 3.74 ×100 copies/μl of BVDV RNA, as no cross-reaction was observed with other viruses. [Conclusion] Overal , the newly established RT-LAMP assay indicates the potential application in both clinical diagnosis and field surveil ance of BVDV.
基金supported by the National Key Research and Development Plan of China[2018YFC1602500]the Natural Science Foundation of Tianjin[19JCZDJC39900]
文摘Objective To establish an ultra-sensitive,ultra-fast,visible detection method for Vibrio parahaemolyticus(VP).Methods We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a(CRISPR/Cas12a)combined with recombinase polymerase amplification and visual detection(CRISPR/Cas12a-VD).Results CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10^(-18)M(single molecule detection)within 30 min without cross-reactivity against other bacteria.When detecting pure cultures of VP,the consistency of results reached 100%compared with real-time PCR.The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10^(2)CFU/g.Conclusion The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods,such as real-time PCR,and has great potential for preventing the spread of pathogens.
基金General Administrationof Quality Supervision, Inspection and Quarantine of China(HK001-2007).
文摘Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications are summarized in this article.
基金Supported by the Guangdong Key S&T Program(2019B020217002)from the Department of Science and Technology of Guangdong Province,China,the Guangdong Poultry Industry Technology System,China(2019KJ128)the earmarked fund for China Agriculture Research System(CARS-41-G16).
文摘Pseudomonas aeruginosa(PA)is an opportunistic pathogen of humans and animals and a common source of nosocomial infections especially of the respiratory tract.Pseudomonas aeruginosa is also a major bacterial disease of poultry and in particular,eggs and newly hatched chicks.In this study,we developed a simple,accurate and rapid molecular detection method using cross priming amplification(CPA)with a nucleic acid test strip to detect P.aeruginosa.The assay efficiently amplified the target gene within 45 min at 62℃only using a simple water bath.The detection limit of the method was 1.18x 102 copiesμL^-1 for plasmid DNA and 4.4 CFU mL^-1 for bacteria in pure culture,and was 100 times more sensitive than conventional PCR.We screened 83 clinical samples from yellow-feather broiler breeder chickens and hospitalized/treated dogs and cats using CPA,PCR and traditional culture methods.The positive sample ratios were 15.3%(13/83)by CPA,13.3%(11/83)by PCR and 12.1%(10/83)by the culture method.The established CPA method has significant advantages for detecting P.aeruginosa.The method is easy to use and possesses high specificity and sensitivity without the requirements of complicated experimental equipment.The PA-CPA assay is especially fit for outdoor and primary medical units and is an ideal system for the rapid detection and monitoring of P.aeruginosa.
