A dual-RPA based lateral flow strip for sensitive,on-site detection of CP4-EPSPS and Cry1Ab/Ac genes in genetically modified crops

Traditional transgenic detection methods require high test conditions and struggle to be both sensitive and efficient.In this study,a one-tube dual recombinase polymerase amplification(RPA)reaction system for CP4-EPSPS and Cry1Ab/Ac was proposed and combined with a lateral flow immunochromatographic assay,named“Dual-RPA-LFD”,to visualize the dual detection of genetically modified(GM)crops.In which,the herbicide tolerance gene CP4-EPSPS and the insect resistance gene Cry1Ab/Ac were selected as targets taking into account the current status of the most widespread application of insect resistance and herbicide tolerance traits and their stacked traits.Gradient diluted plasmids,transgenic standards,and actual samples were used as templates to conduct sensitivity,specificity,and practicality assays,respectively.The constructed method achieved the visual detection of plasmid at levels as low as 100 copies,demonstrating its high sensitivity.In addition,good applicability to transgenic samples was observed,with no cross-interference between two test lines and no influence from other genes.In conclusion,this strategy achieved the expected purpose of simultaneous detection of the two popular targets in GM crops within 20 min at 37°C in a rapid,equipmentfree field manner,providing a new alternative for rapid screening for transgenic assays in the field.

Seismic responses of the steel-strip reinforced soil retaining wall with full-height rigid facing from shaking table test

To investigate the seismic response of the steel-strip reinforced soil retaining wall with fullheight rigid facing in terms of the acceleration in the backfill, dynamic earth pressure in the backfill, the displacements on the facing and the dynamic reinforcement strain distribution under different peak acceleration, a large 1-g shaking table test was performed on a reduced-scale reinforced-earth retaining wall model. It was observed that the acceleration response in non-strip region is greater than that in potential fracture region which is similar with the stability region under small earthquake,while the acceleration response in potential fracture region is greater than that in stability region in middle-upper of the wall under moderately strong earthquakes. The potential failure model of the rigid wall is rotating around the wall toe. It also was discovered that the Fourier spectra produced by the inputting white noises after seismic wave presents double peaks, rather than original single peak, and the frequency of the second peak trends to increase with increasing the PGA(peak ground amplitude) of the excitation which is greater than 0.4 g. Additionally,the non-liner distribution of strip strain along the strips was observed, and the distribution trend was not constant in different row. Soil pressure peak value in stability region is larger than that in potential fracture region. The wall was effective under 0.1 g-0.3 g seismic wave according to the analyses of the facing displacement and relative density. Also, it was discovered that the potential failure surface is corresponds to that in design code, but the area is larger. The results from the study can provide guidance for a more rational design of reinforced earth retaining walls with full-height rigid facing in the earthquake zone.

Strip loading test theory and equipment development

One of the important issues for geotechnical engineers is the characterization of soil properties such as cohesion and internal friction angle by means of soil testing. A new experimental method of soil characterization based on the surface displacement of strip loaded soils is proposed. The theory to relate the soil deformation/displacement to soil strength properties is presented and compared with a series of conventional soil characterization techniques with direct shear tests. The proposed/developed strip loading tests provide reasonably accurate results compared with traditional direct shear tests. The new strip loading physical simulation and testing devices are helpful for understanding soil strength concepts and also provide an effective bridge connecting with engineering mechanics and foundation engineering courses instructions wherein derivation of bearing capacity theory equations is based on the same MohrCoulomb soil strength parameters. The advantages, limitations, and use of the strip loading modeling/testing technique in engineering education and further more in depth researches are discussed in the concluding remarks part.

An Immunochromatographic Strip Test for Rapid Detection of Antibodies against Porcine Circovirus Type 2

Porcine cimovirus type 2 (PCV2) infection causes huge economic losses, but no serological method is available for batch detection of field samples. The aim of the study was to develop a method for large-batch detection of PCV2 infection. Colloidal gold-labeled staphylococcal protein A (SPA) was sprayed on glass fibers to prepare a conjugate pad. The recombinant ORF2 protein of PCV2 was blotted on the test line of the nitrocellulose (NC) membrane, and pig IgG was streaked on the control line of the NC membrane. Then, the immunochromatographic strip was used for detection of antibodies against PCV2. The results show that the strip test was simple and the results could be determined within 10 min with naked eyes. The test strip was highly specific for pig serum against PCV2 and no cross-reaction with pig serum against other pathogens was observed. The test strip had close similarity with ELISA. Storage at RT for 6 months did not affect the specificity and sensitivity obviously. A total of 324 clinical pig sera were detected by both ELISA and the developed test strip, and the coincidence was 98.77%. Therefore, the developed immunochromatographic strip is specific, sensitive, stable, fast and simple, and it is suitable for on-site detection of antibodies against PCV2.

Evaluation of the immunochromatographic strip test for the rapid diagnosis of antenatal syphilis in women in Eldoret,Kenya

Objective： This study compared the performance of the immunochromatographic strip （ICS） to the Venereal Disease Research Laboratory （VDRL） test and Treponema pallidum haemagglutination assay （TPHA） at a primary health care setting. Methods： The study group was comprised of 150 females randomly drawn from a population of pregnant women attending their first antenatal visit or follow-up visits at West Maternity Hospital in Eldoret Kenya, but without a previous syphilis test during that pregnancy. On-site VDRL, ICS and TPHA tests were performed and immediate treatment provided where appropriate. The performance of the three tests was compared, Results： The sero-prevalence of syphilis as determined by the VDRL test was 3%. There was no significant difference between the ICS and the VDRL test （P 〉 0.05）. The sensitivity and specificity of the ICS test were 80% and 98.6% respectively, while the negative predictive value （NPV） and positive predictive value （PPV） were both 100%. On the other hand, the sensitivity and specificity of the VDRL test were 66.7% and 99.3%, while the NPV and PPV were 80% and 98.6% respectively. The Treponema pallidum haemagglutination assay was used as a reference test and had sensitivity, specificity, NPV and PPV of 100%. Conclusion： The diagnostic accuracy of the ICS compared favorably with theVDRL gold standard. The use of the ICS in Kenya can improve the diagnosis of syphilis in health facilities both with and without laboratories and allow community health care workers to make a rapid diagnosis of the disease, and consequently make immediate therapeutic decisions.

''Blue Dream Pregnancy Test Strip''For Detecting Early Pregnancy

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Determination of the Friction Coefficient in the Flat Strip Drawing Test

Current work is focused on the influence of friction in deep drawing process. Friction measurements were also conducted using a modified tribotester based on strip sliding between tools. Four different tool surfaces were tested under similar contact conditions regarding contact area, normal pressure, sliding speed, lubricant and surface characteristics to calculate the friction coefficient between the tool surface and a high strength low alloy steel sheet HSLA 380. The results showed that friction coefficient varies over a wide range with different lubricating conditions and different sliding velocities. For some sliding velocities, the coefficient of friction is stable and low, while for others it is unstable and higher. Results of the experiments reveal that this novel tribotester is a very useful tool to evaluate and compare the friction between steel sheet and tool surfaces in alloyed steel for cold working applications. The outcomes have only small dispersion within the different test series, which indicates a stable process with good repeatability. The test method enables comparison of different surface finishes and treatments, lubricants and coatings in terms of friction and galling under conditions similar to those found in sheet metal forming processes. The four different types of surfaces considered for this study were grinded, polished, nitrided and quenched/tempered. The main difference among the tested tools in this work was the surface roughness, which was found to have a strong influence on friction.

Testing of Anti-stripping Property of Friction Spun Core Yarn

Friction spun core yarn has two components: filament core and staple fiber sheath. Under axial rubbing action, the failure mode of the core yarn is the stripping of the sheath from the core. This paper introduces a method to test the anti - stripping property of the core yarn. With a modified Universal Testing Machine, the stripping resistance of friction spun core yarn can be continuously measured. Some factors Influencing the measurements are discussed in detail. The testing results are compared with those from a Y731 Yarn Abrasion Tester and fur - ther confirmed by weaving practice.

减蛋综合征病毒抗体胶体金免疫层析试纸条的研制及初步应用

为建立快速检测减蛋综合征病毒(EDSV)抗体的方法,基于EDSV的Fiber蛋白,制备快速检测EDSV抗体的免疫层析试纸条,为养禽业提供针对病原微生物的感染预警及免疫评估。利用胶体金标记EDSV Fiber蛋白抗原为探针,将羊抗鸡IgG和抗Fiber蛋白的抗体包被到硝酸纤维膜上,分别作为质控线和检测线,对家禽EDSV抗体水平进行监测。结果表明,制备的EDSV免疫层析试纸条具有高度特异性,与其他家禽病毒抗体无交叉反应,仅与EDSV抗体反应,在试纸条上产生可见的检测线;并且制备的试纸条具有高度稳定性,在室温条件下可至少保存6个月。应用制备的检测EDSV抗体的免疫层析试纸条对从河南、安徽、山东等地养鸡场采集的576份鸡血清样本检测,与血凝抑制试验(HI)结果进行平行比较,二者的Kappa值为0.878,具有良好一致性。综上,制备的检测ESDV抗体的免疫层析试纸条具有高度的特异性、敏感性、稳定性和易操作性,对EDSV抗体的临床检测具有实际应用价值。

基于实车路试的高速公路减速标线运行效果研究

为探寻高速公路减速标线设置对小客车的纵向运行特性的影响,明确减速标线的减速效果,以G50沪渝高速重庆段的3处减速标线区段为对象,开展了实车驾驶试验,采集了自然驾驶状态下的车辆速度、加速度和轨迹等数据,对比分析了车辆经过3处减速标线区段时的速度整体特征、幅值分布和变化模式;同时分析了纵向减速度变化特征;根据速度与加速度的组合示意图和纵向减速标线设置区段内速度的最大降幅、起止点的速度差异性,评价了减速标线的实施效果。结果表明:由于道路线形条件不同,3处减速标线区段的车辆行驶速度整体特征和变化模式存在差异,其中弯坡组合路段车辆速度存在波动,分为“近匀速—减速—加速—稳定—加速”的五阶段变化模式;减速度中位值和85%位值分别为0.123 m/s^(2)、0.225 m/s^(2)、0.185 m/s^(2)和0.346 m/s^(2)、0.513 m/s^(2)、0.489 m/s^(2)。减速标线主要有两方面作用效果:能够使车辆运行车速显著降低,或能够抑制车辆速度增加的趋势。研究成果为高速公路减速标线有效性评价及安全改善提供了基础数据支持。

弗氏柠檬酸杆菌RPA-LFD快速检测方法的建立及应用

为建立一种针对弗氏柠檬酸杆菌(Citrobacter freundii)敏感性高、特异性强的临床快速检测方法,根据该菌定居因子基因cfa的保守序列,设计特异性引物,采用重组酶聚合酶扩增技术(recombinase polymerase amplification,RPA),并结合琼脂糖凝胶电泳(AGE)和侧流层析试纸条(LFD)方法进行反应条件的优化及特异性与灵敏度的检测。结果表明:本研究中所建立的弗氏柠檬酸杆菌重组酶聚合酶扩增结合侧向流动试纸条(RPA-LFD)快速检测方法,在38℃条件下扩增20 min后,能特异性地检测到弗氏柠檬酸杆菌,对弗氏柠檬酸杆菌纯培养物和基因组DNA的最小检出限分别为1.5×102 CFU/mL和200 fg/μL,是常规PCR(cfa引物)检测灵敏度(20 pg/μL)的100倍;利用所建立的RPA-LFD法与常规PCR同时检测杂交鲟攻毒试验样本,两种方法的检测结果一致。研究表明,本研究中建立的弗氏柠檬酸杆菌RPA-LFD方法,操作简便、反应迅速、特异性好、灵敏度高,并且不需要昂贵的仪器,该方法可为未来弗氏柠檬酸杆菌细菌性疾病的早期诊断提供更有效的技术支持。

一种检测砷(Ⅲ)的适配体试纸条

利用三明治夹心法构建基于适配体胶体金的试纸条,目的在于开发切实可行的As(Ⅲ)检测应用装置。通过As(Ⅲ)与金标适配体的特异性结合,将其截留在试纸条检测线处。纳米金作为显色材料,其颗粒直径为(13±1)nm。经过优化反应条件,选择Satorius CN 140作为硝酸纤维素膜,控制线上捕获探针链霉亲和素与生物素的最佳比例为1∶4,对应的划线浓度为10μmol/L。检测线上1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐与N-羟基琥珀酰亚胺的最优比例为1∶3,金标适配体最优浓度为0.5μmol/L。在最佳反应条件下,观察试纸条的显色结果,颜色响应信号与砷浓度形成很好的梯度关系,检测限为0.1μmol/L。本试纸条对As(Ⅲ)具有高度的特异性,并且稳定性良好。其检测成本不足1元,可满足低收入地区普通人群实时检测As(Ⅲ)的要求。

农药残留快速检测试纸条的研制及应用

简单快速地筛查有机磷和氨基甲酸酯类农药残留对食品安全具有重要意义。采用酶抑制法,以重氮固红RC盐为指示剂、乙酸吲哚酚为乙酰胆碱酯酶(AChE)底物,设计开发了一种一步式快速检测试纸条。该试纸条底部为塑料胶板,上面依次粘贴样品垫、酶垫、阻滞垫、底物垫、吸附垫和固定有重氮固红RC盐的硝酸纤维素(NC)膜,通过检测区域颜色的变化可以实现农药残留的定性或定量分析,操作方法简单快捷。不同农药的检出限分别如下:马拉硫磷为0.6 mg·L^(-1),氧化乐果为0.4 mg·L^(-1),乙酰甲胺磷为1.3 mg·L^(-1),敌敌畏为0.1 mg·L^(-1),呋喃丹为0.1 mg·L^(-1),涕灭威为0.07 mg·L^(-1),且在实际果蔬样品检测中显示出良好的重现性和高灵敏度。

E-test试条对异质性耐万古霉素葡萄球菌的检测

目的 探讨E test对异质性耐万古霉素葡萄球菌的检测价值。方法 从连续接触万古霉素的脑心浸液琼脂 (BHIA)平板上挑取万古霉素异质性耐药葡萄球菌菌株 ,应用万古霉素E test试条、药敏纸片和琼脂平板对倍稀释法测定药敏结果。结果 E test检测的对万古霉素异质性耐药葡萄球菌MRSH10 9,MRSH2 ,MRSH13 ,MRSH 3 1,MRSH 60 ,MRSH 10 8,MRSA73 ,MRSA97,MRSA10 2的MIC依次为 :16、2 4、48、48、16、3 2、2 4、2 4mg/L ,其中耐药 7株占 77 8% ( 7/9) ,中介 2株占 2 2 2 % ( 2 /9) ;琼脂对倍稀释法的MIC为 :16、3 2、64、3 2、16、3 2、3 2、16、3 2mg/L ,耐药 6株占 66 7% ( 6/9) ,中介 3株占 3 3 3 % ( 3 /9) ;药敏纸片直径依次为 :14、12、10、11、10、15、13、10 ,13mm ,敏感率为 11 1% ( 1/9)。结论 E test可以便捷直观地鉴定对万古霉素异质性耐药葡萄球菌的药敏结果 ,与琼脂平板对倍稀释法接近 ,优于纸片法。

基于碳系材料的标准应变传感条应用研究

针对传统电阻应变测量过程中,电阻应变片只能适应局部应变的点测,不能进行全域性测量且粘贴工艺复杂的特点,本文提出了采用价格低廉的碳系材料,利用改进凹版印刷技术制备标准长度传感条,采用裁剪及拼接的方法,实现传感条测试区域的任意调整,介绍了基于电阻变化率的传感条应变测试基本原理,通过等截面悬臂梁静载实验考核了标准传感条的各项性能指标,并通过钢筋混凝土弯曲梁实验评价了标准应变传感条针对混凝土梁小应变时全域性的测量效果,实验结果表明:该标准应变传感条具有制备方法简单、组合方式多样、小应变响应跟随性好、适用于户外现场测量的特点,可推广应用于混凝土结构的健康监测领域。

基于蛋白序列比对方法获得灵敏度抗体的诺如病毒免疫层析试剂条的临床应用价值

目的探讨基于蛋白序列比对方法获得灵敏度抗体的诺如病毒免疫层析试剂条的临床应用价值。方法选取150例疑似诺如病毒感染患者作为研究对象,回顾性分析所有患者的临床资料。所有患者均接受常规检查和优化检测试纸的诺如病毒抗原检测。利用患者临床数据对检出结果进行验证,从检出率、重复率、符合率、抗干扰能力和交叉反应等方面对该检测优化方法的检测性能进行评估。结果优化后的抗原检测试剂条的检测准确率高于常规抗原快速检测试剂,差异有统计学意义(P<0.05)。通过评估分析发现,诺如病毒抗原检测中的一些关键因素,如样本采集时间、采集方式、保存方式等对检测结果的影响较大。通过对上述因素进行控制和管理,可以提高诺如病毒抗原检测的准确性和可靠性。结论基于蛋白序列比对方法获得灵敏度抗体的诺如病毒试剂条的临床应用价值较高,可以优化检测方法,提高检测准确性和可靠性,减少漏诊和误诊。

牛诺瓦病毒和牛轮状病毒双重RAA-LFD快速检测方法的建立及应用

为建立能同时检测牛诺瓦病毒(bovine norovirus,BNoV)和牛轮状病毒(bovine rotavirus,BRV)的重组酶辅助扩增-侧流层析试纸条(recombinase aided amplification-lateral flow dipstick,RAA-LFD)快速检测方法。本试验按照RAA引物探针设计原则,分别针对BNoV RdRp基因和BRV VP7基因的保守区设计引物和探针,制备标准重组质粒,建立并优化RAA-LFD反应体系,同时对该检测方法进行特异性、敏感性及重复性评估,用建立的RAA-LFD法与PCR法、qPCR法分别对采集的168份腹泻犊牛的临床样本进行平行检测。结果显示,该方法可在20 min,39℃的条件下扩增目标片段,对BNoV和BRV敏感度均为103 copies·μL^(-1),与PCR的检测结果基本一致,且与牛冠状病毒(bovine coronavirus,BCoV)、牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)和牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)无交叉反应,虽低于qPCR检测结果,但可通过肉眼直接观察试纸条的检测结果。综上所述,RAA-LFD法灵敏度较高、且简便、快速、同时不依赖于仪器、专业操作人员,适用于现场检测。

基于紫胶红素的靶标有色化免疫层析试纸条联检大肠杆菌O157∶H7和沙门氏菌

为摆脱免疫层析试纸条对配对抗体的依赖,文章设计了一种新型靶标有色化免疫层析试纸条,使用紫胶红素和十二合水硫酸铝钾媒染剂,通过先媒后染的方法,实现了免疫层析试纸条的“可视化”。验证结果显示,该试纸条对大肠杆菌O157∶H7和沙门氏菌的检测限均达到了106 CFU/mL;同时,与常见食源性致病菌间不存在交叉反应,在鸡胸肉、鸡蛋、牛奶等食品基质中能够在增菌9 h内检出。文章成功设计了一种操作简单、检测成本低,更易国产化的快速检测免疫层析试纸条,为后续免疫层析试纸条的设计提供了新思路。

小比尺条形基础承载力模型试验的尺寸效应研究

开展小比尺模型试验是土力学承载力研究中的重要方法,但小比尺模型的尺寸效应会影响试验的定量结果,导致小比尺试验一般仅能用于定性研究。在饱和砂土地基中开展了20组条形基础承载力的小比尺模型试验,通过改变基础尺寸、埋深以及砂土的密实度研究承载力的变化,进而引入离散元分析方法,采用抗转动模型模拟砂土颗粒的力学行为,对条形基础承载力试验进行模拟,从微观角度揭示小比尺模型试验尺寸效应的来源和对宏观承载力的影响规律。研究结果表明,饱和砂土地基密实度与基础埋深越大,小比尺实试验的尺寸效应越明显,尺寸效应的产生是应力水平变化、破坏模式改变以及渐进破坏程度综合作用的结果。

环介导等温扩增技术结合乳胶微球试纸条检测副溶血性弧菌

目的建立环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术结合乳胶微球试纸条(latex microsphere test strips,LMTS)快速检测副溶血性弧菌(Vibrio parahaemolyticus,VP)的方法。方法以副溶血性弧菌的不耐热溶血素(TLH)基因作靶标设计引物,6-羧基荧光素(6-carboxyfluorescein,6-FAM)和生物素(Biotin)标记引物,对体系各项反应参数进行优化;将新鲜菌液作10倍梯度稀释后进行LAMP、聚合酶链式反应(polymerase chain reaction,PCR)和实时荧光定量LAMP(quantitative LAMP,qLAMP)反应,比较三者的敏感度;对副溶血性弧菌、大肠埃希氏菌、福氏志贺氏菌、粪链(肠)球菌、单核细胞增生李斯特菌、鼠伤寒沙门氏菌、铜绿假单胞菌进行LAMP扩增,验证其特异性;虾样品用菌液进行模拟污染,分析LAMP-LMTS的可靠性。结果LAMP-LMTS方法的灵敏度可达4.16×10^(2)copies/μL,与qLAMP、LAMP-琼脂糖凝胶电泳(agarose gel electrophoresis,AGE)方法结果一致,检出限要比利用外引物建立的PCR方法低10倍的拷贝数,灵敏度更高。结论该方法检测副溶血性弧菌具有检测时间短、灵敏度高、特异性强、操作简便等特点,可应用于基层实验室、应急检测或现场监测,具有较高的推广价值。