基于病菌孢子捕捉和real-time PCR技术的田间空气中小麦白粉病菌孢子动态监测及病情估计模型研究

利用Burkard定容式孢子捕捉器结合real-time PCR定量技术,分别对种植高抗、中感和高感白粉病小麦品种的田间空气中白粉病菌分生孢子浓度进行监测,结果表明,real-time PCR定量与传统的显微观察计数两种方法测得的孢子浓度呈显著正相关(P≤0.01),且两种病菌孢子计数方法在同一抗性品种上监测到的孢子浓度动态相近。此外,两种方法测得的孢子浓度与各气象因子的相关性分析结果一致,空气中的白粉病菌孢子浓度主要与空气相对湿度显著正相关。在此基础上,利用两种方法测定的田间空气中白粉病菌孢子浓度分别建立了基于累积孢子浓度的田间病情估计模型。分析发现,基于两种孢子浓度测定方法建立的病情估计模型间无显著性差异,表明real-time PCR定量技术测定的孢子浓度在构建白粉病病情估计模型上具有一定可行性。该结果为real-time PCR定量技术与病菌孢子捕捉技术相结合用于小麦白粉病的监测和预测提供理论依据。

基于Real-time PCR法检测乳粉中牛源性成分定量研究

基于Real-timePCR建立了乳粉中牛源性成分相对定量检测方法,并对牛的特异性引物与探针进行了特异性、灵敏度和稳定性测试。通过模拟不同浓度牛乳粉与马乳粉混合样本,根据其△Ct值的函数关系进行线性拟合进而绘制标准曲线,建立乳粉中牛源性成分的相对定量检测。结果显示,该方法的最低检测限为0.00001 mg/mL,回收率为91.11%~119.2%,组间变异系数≤0.58%、组内变异系数≤1.44%。说明该方法在特异性与稳定性上适用于乳粉中牛源性成分及含量的掺假检测。

一种基于real-time PCR技术的TTV检测方法的建立及应用

目的:本研究旨在开发一种具有更高灵敏度和特异性的TTV检测技术,为揭示TTV在多种疾病过程中的作用提供重要的技术支持。方法:为了更精确、灵敏的检测TTV,本研究分析了目前公布的所有亚型的TTV基因序列,在此基础上建立了一种基于UTR区域的real-time PCR检测方法,并与文献报道应用较为广泛的PCR检测方法进行了对比。结果:本研究建立的方法在1×10^(7)~1×10^(1) copies/μL标准品浓度范围内具有良好的线性关系,相关系数为1.000,斜率为-3.446,检测下限为1×10^(1) copies/μL。重复性试验结果显示,组内变异系数为7.22%,表明本方法重复性、稳定性较强。针对30份临床样本,使用本研究建立的real-time PCR检测方法及目前被多个研究所使用的4套引物进行对比。结果表明,本研究所建立的方法灵敏度显著高于文献中报道的4种方法(P<0.01);Sanger测序结果表明,本方法检测出的30份阳性样本均为TTV,检测特异性为100%。结论:本研究采用基于TaqMan探针的real-time PCR检测方法,检测灵敏性高、覆盖基因型范围广,尤其对于TTV病毒载量较低的情况下能够进行定量检测,对于TTV病毒的致病性及作为免疫标志物的应用提供重要的技术支持。

Prevalence and Antibiotic Resistance of Urinary Tract Pathogens, with Molecular Identification of Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp., Using Multiplex Real-Time PCR

Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resistance necessitates updating diagnostic techniques to ensure higher sensitivity and specificity, especially with advancements in science and medicine. This study aimed to evaluate the prevalence of UTIs and antibiotic resistance profiles through urine culture, as well as to identify Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp. in urine samples using a molecular approach with multiplex real-time PCR. From May 3 to July 25, 2023, at the Pietro Annigoni Biomolecular Research Center (CERBA) and Saint Camille Hospital of Ouagadougou (HOSCO), 209 urine samples collected from patients with suspected UTIs were analyzed using both urine culture and multiplex real-time PCR. Among the 209 patients, 52.15% were male and 47.85% female, with an average age of 46.87 ± 21.33 years. Urine cultures revealed an overall UTI prevalence of 23.44%, with a prevalence of 8.13% in men versus 15.31% in women (P = 0.023). The bacterial prevalence rates were as follows: Escherichia coli (12.92%), Klebsiella spp. (7.18%), Enterobacter cloacae (1.44%), Staphylococcus aureus (0.96%), and other bacteria. Klebsiella spp. demonstrated 100% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, while Escherichia coli showed 96.2% and 65.4% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, respectively. PCR analysis of the target bacteria revealed mono-infection prevalence rates of Klebsiella pneumoniae (10.39%), Klebsiella oxytoca (7.79%), and Acinetobacter spp. (7.79%), along with a co-infection prevalence rate of Klebsiella pneumoniae/Acinetobacter spp. (1.30%). This study demonstrated that PCR, with its high sensitivity and specificity, could effectively distinguish Klebsiella pneumoniae from Klebsiella oxytoca and detect Acinetobacter spp. in less than 24 hours—something urine culture alone could not achieve. The relative ease of automating urine PCR testing, combined with its diagnostic accuracy and rapid turnaround time, makes it a valuable addition to modern medical practice for the laboratory diagnosis of UTIs.

24重荧光real-time PCR技术在食物中毒快速检测中的应用

目的:应用24重荧光real-time PCR检测技术快速筛检食物中毒病原菌,结合国家标准中的培养法探讨24重荧光real-time PCR检测技术符合性和应用价值。方法:采用高灵敏度、高特异性的24重荧光real-time PCR检测技术作为中毒病原菌的初筛方法,国标方法进行细菌分离培养,并对分离出的病原菌进行生化鉴定。结果:5份食物中毒患者肛拭子在增菌前检出4份霍乱弧菌核酸(非O1/非O139群,24重荧光real-time PCR),9份患者肛拭子在增菌后检出5株霍乱弧菌(非O1/非O139群,国标培养法),其中包含4份PCR技术初筛阳性样品,两个方法的符合率为80%。所有样本均未检出沙门氏菌、志贺菌、副溶血性弧菌、致泻性大肠埃希菌以及金黄色葡萄球菌。结论:应用24重荧光real-time PCR检测技术同时检测24种常见致病病原菌,能高效锁定中毒病原菌。将其与国标培养法相结合,对临床治疗和食物中毒快速处置能起到积极作用,值得应用和推广。

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

肉中猪源性成分Real-time PCR定量检测技术

【目的】建立一种快速、准确的肉中猪源性成分定量检测方法。【方法】首先从GenBank数据库中筛选猪特异性的微卫星DNA,根据微卫星DNA核酸序列设计引物,对常见10种动物基因组DNA进行PCR扩增,通过有无扩增产物判断筛选的微卫星DNA对猪源性成分的特异性。然后根据微卫星DNA核酸序列,设计特异性引物和探针,建立猪源性成分Real-time PCR检测方法,采用双标准曲线分别对猪源性成分和总动物源性成分进行定量,计算猪源性成分的百分含量。【结果】筛选到猪特异性微卫星DNA(Accession EF172428),根据其序列设计的引物SEQ-sus2-F/R只能从猪基因组DNA中扩增出目的条带,其他动物的基因组均无目的条带扩增。建立的Real-time PCR检测方法灵敏度为0.02 ng/25μL反应体系。该方法能够准确检测出混合DNA样品中猪源性成分和混合肉样品中猪源性成分,百分误差分别约为1.32%和1.06%-7.12%。【结论】本研究利用Real-time PCR技术建立的定量猪源性成分的检测方法可以用来检测猪源性成分在混合样品中的百分含量。

猪NLRP3基因Real-time PCR检测方法的建立及应用

根据NCBI NLRP3 cds(NM-001256770)序列设计1对特异性引物,构建标准品质粒pMD18T-NLRP3-189。通过反应条件优化、稳定性、敏感性等试验,成功建立了NLRP3的SYBR GreenⅠreal-time PCR检测方法,用该方法定量检测猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)感染后的猪肺组织中NLRP3表达水平。最佳引物浓度为0.15μmol/L,建立的标准曲线方程为y=-3.5196x+36.968,E值为0.96,最低检测量为84个拷贝,熔解曲线有单一峰,批内变异系数CV为0.067%~0.184%,批间变异系数为0.221%~0.594%,重复性好。该方法检测仔猪在感染Mhp后肺组织中的NLRP3 mRNA表达水平,感染组与健康组相比差异显著(P<0.01),NLRP3表达水平明显增高,在感染后14 d可达到高峰,平均mRNA拷贝数达18000左右,可持续42 d。表明该检测方法稳定性良好,精确度高,特异性强,为进一步研究猪炎症小体NLRP3的激活及其相关炎症反应机制提供了技术支持。

多主棒孢SdhB-H278Y突变位点real-time PCR检测体系的建立与应用

根据GenBank已登录序列中黄瓜多主棒孢琥珀酸脱氢酶B亚基(SdhB)基因序列差异,针对SdhB-H278Y突变设计特异性引物,建立SdhB-H278Y突变实时荧光定量PCR(real-time PCR)检测体系。结果表明:供试多主棒孢携带SdhBH278Y、SdhB-I280V突变;SdhB-H278Y突变株对啶酰菌胺抗性较强,EC50值为21.47μg·mL^(-1)或>30μg·mL^(-1);建立的real-time PCR检测体系具有良好的线性关系,相关系数R2=0.9929,可特异性检测SdhB-H278Y突变,灵敏度为3.6×10^(-4) ng·μL^(-1),为AS-PCR的10倍。利用携带SdhB-H278Y突变不同比例的基因组DNA对检测体系进行验证,预期值与检测值具有很高的相关性,R^(2)=0.9997;利用该检测体系对山东地区黄瓜棒孢叶斑病病斑中多主棒孢SdhB-H278Y突变株所占比例进行检测,检测结果为0.12%~2.69%。综上,本试验建立的real-time PCR检测体系高效、灵敏、定量,可用于多主棒孢SdhB-H278Y突变的检测,为黄瓜棒孢叶斑病抗性治理提供技术支持。

羊肉中貉源成分Real-time PCR检测方法的建立

[目的]检测羊肉中是否含有貉肉成分。[方法]通过实时荧光定量PCR方法,以cytB为靶基因设计特异性检测引物,选取8个不同物种的肌肉组织样本为研究对象,根据其ΔCt值函数关系进行线性拟合建立标准曲线。[结果]该检测方法所用引物能将貉与其他物种区分,特异性较强,且最低检测限可达到3.2 pg/μL,回收率在97.71%~104.36%,组内变异系数≤0.28%,组间变异系数≤1.08%。[结论]该研究建立的羊肉中貉肉源成分实时荧光定量检测方法具有良好的特异性和敏感性,可用该方法检测实际羊肉样品中是否有貉肉源成分,为羊肉制品掺假的检测提供简单快捷准确的技术手段和执法依据。

A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus,African swine fever virus,and atypical porcine pestivirus

With the implementation of the C-strain vaccine,classical swine fever(CSF) has been under control in China,which is currently in a chronic atypical epidemic situation.African swine fever(ASF) emerged in China in 2018 and spread quickly across the country.It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.Atypical porcine pestivirus(APPV) was first detected in Guangdong Province,China,in 2016,which mainly harms piglets and has a local epidemic situation in southern China.These three diseases have similar clinical symptoms in pig herds,which cause considerable losses to the pig industry.They are difficult to be distinguished only by clinical diagnosis.Therefore,developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.In this study,three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV(5’ UTR),African swine fever virus(ASFV)(B646L),and APPV(5’ UTR),followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.The results showed that the method did not cross-react with other swine pathogens(porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),foot-and-mouth disease virus(FMDV),pseudorabies virus(PRV),porcine parvovirus(PPV),and bovine viral diarrhea virus BVDV).The sensitivity results showed that CSFV,ASFV,and APPV could be detected as low as 1 copy μL–1;the repeatability results showed that the intra-assay and interassay coefficient of variation of ASFV,CSFV,and APPV was less than 1%.Twenty-two virus samples were detected by the multiplex real-time PCR,compared with national standard diagnostic and patented method assay for CSF(GB/T 27540–2011),ASF(GB/T 18648–2020),and APPV(CN108611442A),respectively.The sensitivity of this triple real-time PCR for CSFV,ASFV,and APPV was almost the same,and the compliance results were the same(100%).A total of 451 clinical samples were detected,and the results showed that the positive rates of CSFV,ASFV,and APPV were 0.22% (1/451),1.3%(6/451),and 0%(0/451),respectively.This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV,ASFV,and APPV.

A multifunctional shear apparatus for rocks subjected to true triaxial stress and high temperature in real-time

Deep engineering disasters,such as rockbursts and collapses,are more related to the shear slip of rock joints.A novel multifunctional device was developed to study the shear failure mechanism in rocks.Using this device,the complete shearedeformation process and long-term shear creep tests could be performed on rocks under constant normal stiffness(CNS)or constant normal loading(CNL)conditions in real-time at high temperature and true-triaxial stress.During the research and development process,five key technologies were successfully broken through:(1)the ability to perform true-triaxial compressioneshear loading tests on rock samples with high stiffness;(2)a shear box with ultra-low friction throughout the entire stress space of the rock sample during loading;(3)a control system capable of maintaining high stress for a long time and responding rapidly to the brittle fracture of a rock sample as well;(4)a refined ability to measure the volumetric deformation of rock samples subjected to true triaxial shearing;and(5)a heating system capable of maintaining uniform heating of the rock sample over a long time.By developing these technologies,loading under high true triaxial stress conditions was realized.The apparatus has a maximum normal stiffness of 1000 GPa/m and a maximum operating temperature of 300C.The differences in the surface temperature of the sample are constant to within5C.Five types of true triaxial shear tests were conducted on homogeneous sandstone to verify that the apparatus has good performance and reliability.The results show that temperature,lateral stress,normal stress and time influence the shear deformation,failure mode and strength of the sandstone.The novel apparatus can be reliably used to conduct true-triaxial shear tests on rocks subjected to high temperatures and stress.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Manila Clam Ruditapes philippinarum Under Hypoxic Stress

Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.

Real-time data processing method for CO_(2) dispersion interferometer on EAST

A real-time data processing system is designed for the carbon dioxide dispersion interferometer(CO_(2)-DI)on EAST.The system utilizes the parallel and pipelining capabilities of an fieldprogrammable gate array(FPGA)to digitize and process the intensity of signals from the detector.Finally,the real-time electron density signals are exported through a digital-to-analog converter(DAC)module in the form of analog signals.The system has been successfully applied in the CO_(2)-DI system to provide low-latency electron density input to the plasma control system on EAST.Experimental results of the latest campaign with long-pulse discharges on EAST(2022–2023)demonstrate that the system can respond effectively in the case of rapid density changes,proving its reliability and accuracy for future electron density calculation.

Real-Time Intelligent Diagnosis of Co-frequency Vibration Faults in Rotating Machinery Based on Lightweight-Convolutional Neural Networks

The co-frequency vibration fault is one of the common faults in the operation of rotating equipment,and realizing the real-time diagnosis of the co-frequency vibration fault is of great significance for monitoring the health state and carrying out vibration suppression of the equipment.In engineering scenarios,co-frequency vibration faults are highlighted by rotational frequency and are difficult to identify,and existing intelligent methods require more hardware conditions and are exclusively time-consuming.Therefore,Lightweight-convolutional neural networks(LW-CNN)algorithm is proposed in this paper to achieve real-time fault diagnosis.The critical parameters are discussed and verified by simulated and experimental signals for the sliding window data augmentation method.Based on LW-CNN and data augmentation,the real-time intelligent diagnosis of co-frequency is realized.Moreover,a real-time detection method of fault diagnosis algorithm is proposed for data acquisition to fault diagnosis.It is verified by experiments that the LW-CNN and sliding window methods are used with high accuracy and real-time performance.

Integrated strategy for real-time wind power fluctuation mitigation and energy storage system control

To address the impact of wind-power fluctuations on the stability of power systems,we propose a comprehensive approach that integrates multiple strategies and methods to enhance the efficiency and reliability of a system.First,we employ a strategy that restricts long-and short-term power output deviations to smoothen wind power fluctuations in real time.Second,we adopt the sliding window instantaneous complete ensemble empirical mode decomposition with adaptive noise(SW-ICEEMDAN)strategy to achieve real-time decomposition of the energy storage power,facilitating internal power distribution within the hybrid energy storage system.Finally,we introduce a rule-based multi-fuzzy control strategy for the secondary adjustment of the initial power allocation commands for different energy storage components.Through simulation validation,we demonstrate that the proposed comprehensive control strategy can smoothen wind power fluctuations in real time and decompose energy storage power.Compared with traditional empirical mode decomposition(EMD),ensemble empirical mode decomposition(EEMD),and complete ensemble empirical mode decomposition with adaptive noise(CEEMDAN)decomposition strategies,the configuration of the energy storage system under the SW-ICEEMDAN control strategy is more optimal.Additionally,the state-of-charge of energy storage components fluctuates within a reasonable range,enhancing the stability of the power system and ensuring the secure operation of the energy storage system.

Nanomaterial-assisted wearable glucose biosensors for noninvasive real-time monitoring:Pioneering point-of-care and beyond

This review explores glucose monitoring and management strategies,emphasizing the need for reliable and userfriendly wearable sensors that are the next generation of sensors for continuous glucose detection.In addition,examines key strategies for designing glucose sensors that are multi-functional,reliable,and cost-effective in a variety of contexts.The unique features of effective diabetes management technology are highlighted,with a focus on using nano/biosensor devices that can quickly and accurately detect glucose levels in the blood,improving patient treatment and control of potential diabetes-related infections.The potential of next-generation wearable and touch-sensitive nano biomedical sensor engineering designs for providing full control in assessing implantable,continuous glucose monitoring is also explored.The challenges of standardizing drug or insulin delivery doses,low-cost,real-time detection of increased blood sugar levels in diabetics,and early digital health awareness controls for the adverse effects of injectable medication are identified as unmet needs.Also,the market for biosensors is expected to expand significantly due to the rising need for portable diagnostic equipment and an ever-increasing diabetic population.The paper concludes by emphasizing the need for further research and development of glucose biosensors to meet the stringent requirements for sensitivity and specificity imposed by clinical diagnostics while being cost-effective,stable,and durable.

Real-Time Detection and Instance Segmentation of Strawberry in Unstructured Environment

The real-time detection and instance segmentation of strawberries constitute fundamental components in the development of strawberry harvesting robots.Real-time identification of strawberries in an unstructured envi-ronment is a challenging task.Current instance segmentation algorithms for strawberries suffer from issues such as poor real-time performance and low accuracy.To this end,the present study proposes an Efficient YOLACT(E-YOLACT)algorithm for strawberry detection and segmentation based on the YOLACT framework.The key enhancements of the E-YOLACT encompass the development of a lightweight attention mechanism,pyramid squeeze shuffle attention(PSSA),for efficient feature extraction.Additionally,an attention-guided context-feature pyramid network(AC-FPN)is employed instead of FPN to optimize the architecture’s performance.Furthermore,a feature-enhanced model(FEM)is introduced to enhance the prediction head’s capabilities,while efficient fast non-maximum suppression(EF-NMS)is devised to improve non-maximum suppression.The experimental results demonstrate that the E-YOLACT achieves a Box-mAP and Mask-mAP of 77.9 and 76.6,respectively,on the custom dataset.Moreover,it exhibits an impressive category accuracy of 93.5%.Notably,the E-YOLACT also demonstrates a remarkable real-time detection capability with a speed of 34.8 FPS.The method proposed in this article presents an efficient approach for the vision system of a strawberry-picking robot.

A real-time performance improvement method for composite time scale

The composite time scale(CTS)provides a stable,accurate,and reliable time scale for modern society.The improvement of CTS’s real-time performance will improve its stability,which strengths related applications’performance.Aiming at this goal,a method achieved by determining the optimal calculation interval and accelerating adjustment stage is proposed in this paper.The determinants of the CTS’s calculation interval(characteristics of the clock ensemble,the measurement noise,the time and frequency synchronization system’s noise and the auxiliary output generator noise floor)are studied and the optimal calculation interval is obtained.We also investigate the effect of ensemble algorithm’s initial parameters on the CTS’s adjustment stage.A strategy to get the reasonable initial parameters of ensemble algorithm is designed.The results show that the adjustment stage can be finished rapidly or even can be shorten to zero with reasonable initial parameters.On this basis,we experimentally generate a distributed CTS with a calculation interval of 500 s and its stability outperforms those of the member clocks when the averaging time is longer than1700 s.The experimental result proves that the CTS’s real-time performance is significantly improved.

Models to Simulate Effective Coverage of Fire Station Based on Real-Time Travel Times

In recent years,frequent fire disasters have led to enormous damage in China.Effective firefighting rescues can minimize the losses caused by fires.During the rescue processes,the travel time of fire trucks can be severely affected by traffic conditions,changing the effective coverage of fire stations.However,it is still challenging to determine the effective coverage of fire stations considering dynamic traffic conditions.This paper addresses this issue by combining the traveling time calculationmodelwith the effective coverage simulationmodel.In addition,it proposes a new index of total effective coverage area(TECA)based on the time-weighted average of the effective coverage area(ECA)to evaluate the urban fire services.It also selects China as the case study to validate the feasibility of the models,a fire station(FS-JX)in Changsha.FS-JX station and its surrounding 9,117 fire risk points are selected as the fire service supply and demand points,respectively.A total of 196 simulation scenarios throughout a consecutiveweek are analyzed.Eventually,1,933,815 sets of valid sample data are obtained.The results showed that the TECA of FS-JX is 3.27 km^(2),which is far below the standard requirement of 7.00 km^(2) due to the traffic conditions.The visualization results showed that three rivers around FS-JX interrupt the continuity of its effective coverage.The proposed method can provide data support to optimize the locations of fire stations by accurately and dynamically determining the effective coverage of fire stations.