4种植物源性成分多重real-time PCR检测方法的建立及其在食用淀粉中的应用

建立一种可同时快速检测红薯、木薯、马铃薯、玉米源性成分的多重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法。分别以红薯g3pdh基因、木薯g3pdh基因、马铃薯UGPase基因、玉米zSSIIb基因为靶基因设计特异性引物和TaqMan探针,以18S rRNA基因为内参基因,建立多重real-time PCR方法,开展方法学验证,并对不同掺入比例模拟样品和实际淀粉样品进行检测。结果显示,该方法具有高通量、特异性强、灵敏度高等优点。与15种非目标源性均无交叉反应;对目标DNA的检测灵敏度可达到3×10^(-3) ng/μL,且具有良好的线性关系和扩增效率;对淀粉样品的检出限可达0.1%,对50份实际样品进行检测,结果与参比方法一致,说明建立的多重real-time PCR法可用于食用淀粉种类掺假鉴别检测。

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。

马乳酒样乳杆菌马乳酒样亚种real-time PCR检测方法的建立与应用

本研究建立了一种特异性实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)检测方法,根据模式菌株马乳酒样乳杆菌马乳酒样亚种ZW3的16S rDNA序列和全基因组序列设计筛选特异性引物,采用SYBR Green I荧光染料建立real-time PCR方法,并对方法的特异性、灵敏度、重复性和混合体系等进行检测。结果表明,本研究所建立的方法特异性强、灵敏度高、重复性好,建立real-time PCR的标准曲线,其决定系数R2为0.965,具有良好的线性关系,且在马乳酒样乳杆菌马乳酒样亚种及混合体系中可以特异性检出。综上,本研究建立的real-time PCR法可以快速、准确地检测马乳酒样乳杆菌马乳酒样亚种,为马乳酒样乳杆菌的特异性定性定量检测提供了一种新的方法。

Real-time Rescue Target Detection Based on UAV Imagery for Flood Emergency Response

Timely acquisition of rescue target information is critical for emergency response after a flood disaster.Unmanned Aerial Vehicles(UAVs)equipped with remote sensing capabilities offer distinct advantages,including high-resolution imagery and exceptional mobility,making them well suited for monitoring flood extent and identifying rescue targets during floods.However,there are some challenges in interpreting rescue information in real time from flood images captured by UAVs,such as the complexity of the scenarios of UAV images,the lack of flood rescue target detection datasets and the limited real-time processing capabilities of the airborne on-board platform.Thus,we propose a real-time rescue target detection method for UAVs that is capable of efficiently delineating flood extent and identifying rescue targets(i.e.,pedestrians and vehicles trapped by floods).The proposed method achieves real-time rescue information extraction for UAV platforms by lightweight processing and fusion of flood extent extraction model and target detection model.The flood inundation range is extracted by the proposed method in real time and detects targets such as people and vehicles to be rescued based on this layer.Our experimental results demonstrate that the Intersection over Union(IoU)for flood water extraction reaches an impressive 80%,and the IoU for real-time flood water extraction stands at a commendable 76.4%.The information on flood stricken targets extracted by this method in real time can be used for flood emergency rescue.

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.

A Comparative Study between Landmark Based and Real Time Ultrasound Guided Sub Arachnoid Block

Background: Sub arachnoid block (SAB) performed by traditional landmark palpation technique can be inaccurate. This problem is exacerbated by altered patient anatomy due to obesity and age-related changes. A pre-procedural ultrasound scan of the lumbar spine has been shown to be of benefit in guiding lumbar epidural insertion in obstetric patients. Information on the use of real-time ultrasound (RUS) guided SAB, to date, been limited. This study compared RUS guided SAB to traditional landmark guided technique in patients undergoing spinal anesthesia for different surgical procedures. Methods: This was a prospective, single center, comparative observational study conducted in the department of anesthesiology at our center. 560 patients who underwent spinal anesthesia either by landmark based technique or real-time ultrasound-guided methods. The primary outcome was the first attempt success rate of dural puncture when employing the two methods. Results: Baseline characteristics were similar in the two study groups. The first attempt success rate of dural puncture in landmark guided group was 64.3% compared to 72.6% in the ultrasound guided group. This difference was not statistically significant. The procedure performance time was significantly shorter with landmark palpation compared to use of real-time ultrasound guided method. Conclusion: Use of RUS-guided technique does not significantly improve the first attempt success rate of SAB dural puncture during spinal anesthesia compared to the traditional landmark-guided technique.

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。

Real-Time Monitoring of Meteorological Data at In-Situ GCW Remediation Sites

To optimize the self-organization network, self-adaptation, real-time monitoring, remote management capability, and equipment reuse level of the meteorological station supporting the portable groundwater circulation wells, and to provide real-time and effective technical services and environmental data support for groundwater remediation, a real-time monitoring system design of the meteorological station supporting the portable groundwater circulation wells based on the existing equipment is proposed. A variety of environmental element information is collected and transmitted to the embedded web server by the intelligent weather transmitter, and then processed by the algorithm and stored internally, displayed locally, and published on the web. The system monitoring algorithm and user interface are designed in the CNWSCADA development environment to realize real-time processing and analysis of environmental data and monitoring, control, management, and maintenance of the system status. The PLC-controlled photovoltaic power generating panels and lithium battery packs are in line with the concept of energy saving and emission reduction, and at the same time, as an emergency power supply to guarantee the safety of equipment and data when the utility power fails to meet the requirements. The experiment proves that the system has the characteristics of remote control, real-time interaction, simple station deployment, reliable operation, convenient maintenance, and green environment protection, which is conducive to improving the comprehensive utilization efficiency of various types of environmental information and providing reliable data support, theoretical basis and guidance suggestions for the research of groundwater remediation technology and its disciplines, and the research and development of the movable groundwater cycling well monitoring system.

垂体后叶粉中3种动物源性成分多重real-time PCR检测方法的建立及应用

目的基于三重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)技术同时快速检测垂体后叶粉中的猪、牛、羊性动物源性成分。方法采用CTAB法提取样品及对照品DNA,以线粒体基因为靶基因设计并合成三重real-time PCR的引物探针,并对建立方法进行验证,用于检测垂体后叶粉样品是否与标识一致。结果猪、牛、羊源性成分的最低检测浓度为10 pg/μL,采用建立的方法检测3批垂体后叶粉样品,均只检测出猪源性成分。结论建立的方法可用于同时检测垂体后叶粉中的猪、牛、羊源性成分,可为相关工作提供技术支持。

A CNN-Based Single-Stage Occlusion Real-Time Target Detection Method

Aiming at the problem of low accuracy of traditional target detection methods for target detection in endoscopes in substation environments, a CNN-based real-time detection method for masked targets is proposed. The method adopts the overall design of backbone network, detection network and algorithmic parameter optimisation method, completes the model training on the self-constructed occlusion target dataset, and adopts the multi-scale perception method for target detection. The HNM algorithm is used to screen positive and negative samples during the training process, and the NMS algorithm is used to post-process the prediction results during the detection process to improve the detection efficiency. After experimental validation, the obtained model has the multi-class average predicted value (mAP) of the dataset. It has general advantages over traditional target detection methods. The detection time of a single target on FDDB dataset is 39 ms, which can meet the need of real-time target detection. In addition, the project team has successfully deployed the method into substations and put it into use in many places in Beijing, which is important for achieving the anomaly of occlusion target detection.

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.

A step to the decentralized real-time timekeeping network

The composite time scale(CTS) provides an accurate and stable time-frequency reference for modern science and technology. Conventional CTS always features a centralized network topology, which means that the CTS is accompanied by a local master clock. This largely restricts the stability and reliability of the CTS. We simulate the restriction and analyze the influence of the master clock on the CTS. It proves that the CTS's long-term stability is also positively related to that of the master clock, until the region dominated by the frequency drift of the H-maser(averaging time longer than ~10~5s).Aiming at this restriction, a real-time clock network is utilized. Based on the network, a real-time CTS referenced by a stable remote master clock is achieved. The experiment comparing two real-time CTSs referenced by a local and a remote master clock respectively reveals that under open-loop steering, the stability of the CTS is improved by referencing to a remote and more stable master clock instead of a local and less stable master clock. In this way, with the help of the proposed scheme, the CTS can be referenced to the most stable master clock within the network in real time, no matter whether it is local or remote, making democratic polycentric timekeeping possible.

Cyber Resilience through Real-Time Threat Analysis in Information Security

This paper examines how cybersecurity is developing and how it relates to more conventional information security. Although information security and cyber security are sometimes used synonymously, this study contends that they are not the same. The concept of cyber security is explored, which goes beyond protecting information resources to include a wider variety of assets, including people [1]. Protecting information assets is the main goal of traditional information security, with consideration to the human element and how people fit into the security process. On the other hand, cyber security adds a new level of complexity, as people might unintentionally contribute to or become targets of cyberattacks. This aspect presents moral questions since it is becoming more widely accepted that society has a duty to protect weaker members of society, including children [1]. The study emphasizes how important cyber security is on a larger scale, with many countries creating plans and laws to counteract cyberattacks. Nevertheless, a lot of these sources frequently neglect to define the differences or the relationship between information security and cyber security [1]. The paper focus on differentiating between cybersecurity and information security on a larger scale. The study also highlights other areas of cybersecurity which includes defending people, social norms, and vital infrastructure from threats that arise from online in addition to information and technology protection. It contends that ethical issues and the human factor are becoming more and more important in protecting assets in the digital age, and that cyber security is a paradigm shift in this regard [1].

Effects of data smoothing and recurrent neural network(RNN)algorithms for real-time forecasting of tunnel boring machine(TBM)performance

Tunnel boring machines(TBMs)have been widely utilised in tunnel construction due to their high efficiency and reliability.Accurately predicting TBM performance can improve project time management,cost control,and risk management.This study aims to use deep learning to develop real-time models for predicting the penetration rate(PR).The models are built using data from the Changsha metro project,and their performances are evaluated using unseen data from the Zhengzhou Metro project.In one-step forecast,the predicted penetration rate follows the trend of the measured penetration rate in both training and testing.The autoregressive integrated moving average(ARIMA)model is compared with the recurrent neural network(RNN)model.The results show that univariate models,which only consider historical penetration rate itself,perform better than multivariate models that take into account multiple geological and operational parameters(GEO and OP).Next,an RNN variant combining time series of penetration rate with the last-step geological and operational parameters is developed,and it performs better than other models.A sensitivity analysis shows that the penetration rate is the most important parameter,while other parameters have a smaller impact on time series forecasting.It is also found that smoothed data are easier to predict with high accuracy.Nevertheless,over-simplified data can lose real characteristics in time series.In conclusion,the RNN variant can accurately predict the next-step penetration rate,and data smoothing is crucial in time series forecasting.This study provides practical guidance for TBM performance forecasting in practical engineering.

Storage time affects the level and diagnostic efficacy of plasma biomarkers for neurodegenerative diseases

Several promising plasma biomarker proteins,such as amyloid-β(Aβ),tau,neurofilament light chain,and glial fibrillary acidic protein,are widely used for the diagnosis of neurodegenerative diseases.However,little is known about the long-term stability of these biomarker proteins in plasma samples stored at-80°C.We aimed to explore how storage time would affect the diagnostic accuracy of these biomarkers using a large cohort.Plasma samples from 229 cognitively unimpaired individuals,encompassing healthy controls and those experiencing subjective cognitive decline,as well as 99 patients with cognitive impairment,comprising those with mild cognitive impairment and dementia,were acquired from the Sino Longitudinal Study on Cognitive Decline project.These samples were stored at-80°C for up to 6 years before being used in this study.Our results showed that plasma levels of Aβ42,Aβ40,neurofilament light chain,and glial fibrillary acidic protein were not significantly correlated with sample storage time.However,the level of total tau showed a negative correlation with sample storage time.Notably,in individuals without cognitive impairment,plasma levels of total protein and tau phosphorylated protein threonine 181(p-tau181)also showed a negative correlation with sample storage time.This was not observed in individuals with cognitive impairment.Consequently,we speculate that the diagnostic accuracy of plasma p-tau181 and the p-tau181 to total tau ratio may be influenced by sample storage time.Therefore,caution is advised when using these plasma biomarkers for the identification of neurodegenerative diseases,such as Alzheimer's disease.Furthermore,in cohort studies,it is important to consider the impact of storage time on the overall results.

Real-time PCR和PCR方法快速检测犬细小病毒

为适应出入境口岸对进出境宠物快速检疫的需要,本研究在建立PCR方法检测犬细小病毒(CPV)的基础上,进一步采用Taqman探针技术建立了快速检测CPV的Real-time PCR方法。通过灵敏度对比试验,证实Real-time PCR方法比PCR方法检测灵敏度显著提高。通过对大量不同采样部位样品的检测证实,本研究建立的Real-time PCR和PCR方法具有较高的可靠性,并可显著提高CPV的阳性检出率。

基因Ⅰ型草鱼呼肠孤病毒TaqMan Real-Time PCR检测方法的建立及应用

为了建立和应用可检测基因I型草鱼呼肠孤病毒（GCRV）的荧光定量检测方法，实验根据基因I型GCRV的s6保守序列，分别设计扩增目的条带661bp普通引物（P1、P2）、扩增目的条带159bp荧光定量引物（F1、F2）和一条探针；同时构建含有目的片段的PVAX1-S6作为标准质粒，10倍梯度稀释构建质粒拷贝数与Ct值的标准曲线（Y=-3．389X＋38．076）。结果表明，此方法在3．9×10^7～3．9×10^1拷贝／uL之间相关性较好，R2为0．992，最小检测量为4拷贝／uL；且与基因Ⅱ型、Ⅲ型GCRV以及其他病原核酸无交叉反应。运用该方法检测16份草鱼出血病疑似样品，阳性结果为4份；而普通PCR检测仅2份显示阳性。本研究建立了针对基因I型GCRV的荧光定量检测方法，具有高效、特异、灵敏、可重复性强的优点，适合于目前基因I型GCRV的临床快速检测和病毒定量分析。

金黄色葡萄球菌基因表达的DNA扣除法Real-time RT PCR相对定量分析

【目的】金黄色葡萄球菌作为一种分布广泛的致病微生物和研究革兰氏阳性菌遗传背景的模式菌株,利用real-time RT PCR对相关毒素及调控基因进行表达定量分析,在生物、医学、食品检测等领域具有较大研究价值。【方法】对制备好的反转录(RT,含有cDNA和DNA)和非反转录(RT—,仅含DNA)样品进行Real-time PCR检测,根据经典(1+E)—△△Ct相对定量算法并结合PCR效率公式建立一种基因表达相对定量分析的DNA扣除法,将得到的Ct值转换为各样品含量,从RT样品中扣除RT—样品的量,无需DNaseⅠ酶解处理就可以去除DNA的影响,RT—样品的检测结果还可同时作为稳定的DNA内参。【结果】采用以上方法分析金黄色葡萄球菌肠毒素A基因(sea)、16S rRNA和RNAⅢ的表达情况,在含有葡萄糖的NB培养基中sea的相对转录水平随着葡萄糖浓度的增大而升高,RNAⅢ的相对转录水平随葡萄糖浓度的变化而产生小幅度的波动,16S rRNA在菌体生长初期时的表达量较为稳定;与绝对定量法比较,结果差异较小(均小于15%),且差异不显著(p>0.05)。【结论】这种基于DNA扣除法的Real-time RT PCR相对定量方法可以有效的对金黄色葡萄球菌的基因表达进行分析。