Redesigned Duplex RT-qPCR for the Detection of GI and GII Human Noroviruses

Human noroviruses(HuNoVs)are major foodborne pathogens that cause nonbacterial acute gastroenteritis worldwide.As the tissue-culture system for HuNoVs is not mature enough for routine detection of the virus,detection is mainly dependent on molecular approaches such as reverse transcription polymerase chain reaction(RT-PCR)and reverse transcription quantitative real-time polymerase chain reaction(RTqPCR).The widely used primers and probes for RT-qPCR were established in the early 2000s.As HuNoVs are highly variant viruses,viral genome mutations result in previously designed primers and/or probes that were perfectly matched working less efficiently over time.In this study,a new duplex RT-qPCR(ND-RT-qPCR)was designed for the detection of genogroup Ⅰ(GⅠ)and genogroup Ⅱ(GⅡ)HuNoVs based on an analysis of viral sequences added in the database after 2010.Using long transcribed viral RNAs,the results demonstrate that the sensitivity of ND-RT-qPCR is as low as one genomic copy for both GⅠ and GⅡ HuNoVs.The performance of ND-RT-qPCR was further evaluated by a comparison with the commonly used Kageyama primer/probe sets for RT-qPCR(Kageyama RT-qPCR)for 23 HuNoV-positive clinical samples.All five GⅠ samples were registered as positive by ND-RT-qPCR,whereas only two samples were registered as positive by Kageyama RT-qPCR.All 18 GⅡ samples were registered as positive by ND-RT-qPCR,while 17 samples were registered as positive by Kageyama RT-qPCR.The sensitivity reflected by the quantification cycle(Cq)value was lower in ND-RT-qPCR than in Kageyama RT-qPCR.Our data suggest that ND-RT-qPCR could be a good fit for the detection of current strains of HuNoVs.

PEDV、PoRVA和PDCoV TaqMan三重RT-qPCR检测方法的建立与初步应用

旨在建立一种可快速同时检测猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)、猪A群轮状病毒(porcine rotavirus type A, PoRVA)和猪丁型冠状病毒(porcine deltacoronavirus, PDCoV)三重荧光定量PCR方法。针对PEDV-N、PoRVA-VP6和PDCoV-M基因设计了引物和探针,条件优化后进行性能评估,并与商品化试剂盒检测对比。结果显示:本研究建立的三重RT-qPCR方法具有良好特异性,对PRRSV、PCV_2和PRV等阳性核酸不发生扩增;具有较高的敏感性,PEDV、PoRVA和PDCoV的最低检测限均达1 copies·μL^(-1);重复性良好,组内和组间变异系数均小于1%;样本适应性广,检测不同样本类型时,变异系数均小于1%。与商品化试剂盒对比,本研究建立的方法PEDV和PoRVA的检测符合率为92.5%和97.5%,并对PDCoV的检测范围更广,对其变异毒株仍有较好的检测效果。本研究建立的检测方法具有特异性好、敏感性高、稳定性强和样本适应性广等优势,为临床腹泻样本提供了一种好的检测方法。

含内参的登革和寨卡病毒三重RT-qPCR检测方法的建立与评估

目的 本研究旨在建立一种登革病毒以及寨卡病毒并含人类基因为内参的三重RT-qPCR检测方法,可以同时检测出内参、登革病毒以及寨卡病毒。方法 针对登革病毒4种血清型的保守区域和寨卡病毒的NS1基因以及人类各个组织中稳定表达的β-actin基因,设计3组特异性引物和探针。构建4种血清型的登革病毒、寨卡病毒以及β-actin标准质粒作为阳性质控品,采用L_(9)(3^(4))正交实验方法确定最佳反应条件,对其特异性、灵敏性及覆盖面进行验证与临床评估,并与检测登革病毒的商品化试剂盒进行了一致性评估。结果 本实验建立的三重RT-qPCR检测方法与12种相近虫媒病毒无非特异性交叉反应;对登革病毒与寨卡病毒的检测灵敏度分别为2.99和2.18 copies/μL组内和组间重复性变异系数均在1.5%以内;与商品化试剂盒相比较,该检测方法对13株登革流行病毒均可获阳性结果;经过Bland-Altman一致性分析,商品化试剂盒和该方法对临床阳性样本检测结果一致性达到92.59%。结论 本研究建立了一种特异性强、灵敏度高的含内参的登革病毒和寨卡病毒三重RT-qPCR检测方法,可作为登革病毒与寨卡病毒感染者的早期快速鉴别诊断的有效工具,也可用于病毒暴发地区的快速筛查。

伪狂犬病病毒RT-qPCR检测方法的建立与应用

为建立伪狂犬病病毒(PRV)高灵敏度、超快速双重实时荧光定量PCR检测方法,根据PRV经典毒株gD和gE基因的相对保守序列设计了多组引物探针,经试验筛选出2组最优特异性引物探针,通过正交试验对反应体系中引物和探针的浓度进行优化,通过特异性、灵敏度和重复性试验评测所建立检测方法的性能.结果表明,建立的检测方法具有多重优点:高灵敏度,gD和gE基因最低检测灵敏度分别为1.06 copies/μL和1.68 copies/μL;超快速,检测时间可缩短至1 h内;特异性好,阴性对照组中常见猪患病毒均无交叉反应,PRV野毒株出现了双gD和gE基因扩增曲线,缺失gE基因的疫苗株仅出现gD基因扩增曲线;重复性强,3次重复性批内与批间试验的变异系数均低于3%.综上所述,本研究建立的双重RT-qPCR检测方法可用于PRV野毒株和疫苗株感染的快速诊断鉴别,为猪伪狂犬病(PR)的诊断及流行病学监测提供了一种快速准确的检测方法.

Electromagnetic responses on microstructures of duplex stainless steels based on 3D cellular and electromagnetic sensor finite element models

Microstructures determine mechanical properties of steels,but in actual steel product process it is difficult to accurately control the microstructure to meet the requirements.General microstructure characterization methods are time consuming and results are not rep-resentative for overall quality level as only a fraction of steel sample was selected to be examined.In this paper,a macro and micro coupled 3D model was developed for nondestructively characterization of steel microstructures.For electromagnetic signals analysis,the relative permeability value computed by the micro cellular model can be used in the macro electromagnetic sensor model.The effects of different microstructure components on the relative permeability of duplex stainless steel(grain size,phase fraction,and phase distribu-tion)were discussed.The output inductance of an electromagnetic sensor was determined by relative permeability values and can be val-idated experimentally.The findings indicate that the inductance value of an electromagnetic sensor at low frequency can distinguish dif-ferent microstructures.This method can be applied to real-time on-line characterize steel microstructures in process of steel rolling.

Progress in weldability research of duplex stainless steels

Duplex stainless steels(DSSs)show better corrosion resistance with higher strength than traditional austenite stainless steels in many aggressive environments,and can be welded properly with almost every welding processes,if proper heat input is provided.Progresses of research works on weldability of DSSs in recent years are reviewed in this paper.Balance control of ferrite/austenite phases is most important for DSSs welding.The phases balance can be controlled with filler materials,nitrogen addition in shielding gas,heat input,post weld heat treatment,and alternating magnetic field.Too high cooling rate results in not only extra ferrite,but also chromium nitride precipitation.While too low cooling rate or heating repeatedly results in precipitation of secondary austenite and intermetallic compounds.In both situations,mechanical properties and corrosion resistance of the DSS joints deteriorate.Recommended upper and lower limits of heat input and maximum interpass temperature should be observed.

猪传染性胃肠炎的RT-qPCR检测方法及防治措施

猪传染性胃肠炎是一种在仔猪中流行率较高的病毒性接触传染病,发病后常导致大量哺乳仔猪死亡,给养猪场(户)带来严重的经济损失。由于该病传播速度快,如不采取有效防范措施,势必对整个养殖业造成严重危害。因此,需要明确猪传染性胃肠炎的流行病学特征,做到科学、有效地识别和诊断,结合临床症状制定针对性治疗方案,确保在短时间内缓解症状,提高治愈率。本研究探讨了猪传染性胃肠炎的RT-qPCR检测与防治措施,希望对广大养猪场(户)有所帮助。

基于转录组测序和RT-qPCR技术的烟草糖酯合成基因挖掘

为挖掘调控烟草糖酯合成的功能基因,采用转录组测序对烟草腺毛细胞和叶细胞的差异表达基因及其功能进行分析,并利用RT-qPCR对分析结果进行验证。结果表明:共获得11962个腺毛细胞和叶细胞的差异表达基因,其中5145个上调表达基因,6817个下调表达基因;上调表达基因主要包括生物学进程(2265个)、分子功能(1169个)、细胞成分(363个)3大类,可将基因序列定位到122条代谢通路中,其中与糖酯合成相关的糖酵解/糖异生、淀粉和蔗糖代谢、苯丙烷类生物合成被高度富集,腺毛细胞中酰基糖通路显著上调,有12个基因在功能上与酰基转移酶有关;在腺毛细胞中,分析得到的12个基因中有11个基因的表达量都高于叶细胞,其中gene63826、gene16194和gene37511表达分别上调195.79倍、166.23倍和132.65倍。上述11个基因可能参与调控腺毛细胞合成糖酯,为下一步验证基因功能提供依据。

用RT-qPCR方法评价4个不同猪品种骨骼肌候选内参基因

【目的】寻找合适的规范化策略研究猪骨骼肌相关基因。【方法】筛选TATA结合蛋白(TBP)、次黄嘌呤磷酸核糖基转移酶(HPRT)、核糖体蛋白L4(RPL4)、肽基脯氨酸异构酶A(PPIA)、β-2微球蛋白(B2M)、酪氨酸3-单加氧酶/色氨酸5-单加氧酶活化蛋白(YWHAZ)、β-肌动蛋白(ACTB)和甘油醛-3-磷酸脱氢酶(GAPDH)8个候选内参基因,选取国外引进的长白、约克与杜洛克公猪,进行三元杂交生产的商品猪(DLY),长白、约克猪进行二元杂交的商品猪(LY),成华猪和藏猪共4个品种进行基因表达正常化评价。采用geNorm、NormFinder和BestKeeper3种常用算法评估候选内参基因的稳定性。将3种常用算法的结果进行综合排名。【结果】8个候选内参基因在不同猪种中表现出较高的表达稳定性,但表达循环阈值不同。【结论】运用3种不同的评估算法可以筛选出相似的候选内参基因,该结果可为猪骨骼肌基因表达RT-qPCR分析内参基因的选择提供参考。

Developing a duplex ARMS-qPCR method to differentiate genotype Ⅰ and Ⅱ African swine fever viruses based on their B646L genes

Africanswinefever(ASF),causedbythe African swine fever virus (ASFV), is an acute, hemorrhagic, and contagious disease of domestic pigs and wild boars.The disease is notifiable and listed by the World Organization for Animal Health (WOAH)(Wang N et al. 2019).

Joint Flexible Duplexing and Power Allocation with Deep Reinforcement Learning in Cell-Free Massive MIMO System

Network-assisted full duplex(NAFD)cellfree(CF)massive MIMO has drawn increasing attention in 6G evolvement.In this paper,we build an NAFD CF system in which the users and access points(APs)can flexibly select their duplex modes to increase the link spectral efficiency.Then we formulate a joint flexible duplexing and power allocation problem to balance the user fairness and system spectral efficiency.We further transform the problem into a probability optimization to accommodate the shortterm communications.In contrast with the instant performance optimization,the probability optimization belongs to a sequential decision making problem,and thus we reformulate it as a Markov Decision Process(MDP).We utilizes deep reinforcement learning(DRL)algorithm to search the solution from a large state-action space,and propose an asynchronous advantage actor-critic(A3C)-based scheme to reduce the chance of converging to the suboptimal policy.Simulation results demonstrate that the A3C-based scheme is superior to the baseline schemes in term of the complexity,accumulated log spectral efficiency,and stability.

High throughput screening of localised and general corrosion in type 2205 duplex stainless steel at ambient temperature

Bipolar electrochemistry is used to produce a linear potential gradient across a bipolar electrode(BPE),providing direct access to the anodic and cathodic reactions under a wide range of applied potentials.The occurrence of pitting corrosion,crevice corrosion,and general corrosion on type 2205 duplex stainless steel(DSS 2205)BPE has been observed at room temperature.The critical pit depth of 10-20μm with a55%-75% probability of pits developing into stable pits at potential from+0.9 to+1.2 V vs.OCP(open circuit potential)are measured.All pit nucleation sites are either within ferritic grains or at the interface between austenite and ferrite.The critical conditions for pitting and crevice corrosion are discussed with Epit(critical pitting potential)and Ecre(critical crevice potential)decreasing from 0.87 and 0.80 V vs.OCP after150 s of exposure to 0.84 and 0.76 V vs.OCP after 900 s of exposure,respectively.Pit growth kinetics under different applied bipolar potentials and exposure times have been obtained.The ferrite is shown to be more susceptible to general dissolution.

In Band Full Duplex (IBFD) Technology for Next Generation Wireless Networks: A Survey in Cellular Networks

Next-Generation(NextG)wireless communication networks with their widespread applications require high data rates,seamless connectivity and high quality of service(QoS).To cope up with an unprecedented rise of data hungry applications,users demand more spectral resources imposing a limitation on available wireless spectrum.One of the potential solutions to address the spectrum scarce issue is to incorporate in band full duplex(IBFD)or full duplex(FD)paradigm in next generation networks including 5G new radio(NR).Recently,FD has gained the research interest in cellular networks for its potential to double the wireless link capacity and enhancing spectral efficiency(SE).In half duplex(HD)cellular networks,base stations(BSs)can either perform uplink(UL)or downlink(DL)transmission at a particular time instant leading to reduced throughput levels.Due to the advancement in the self interference reduction(SIR)techniques,full duplex base stations(FD-BSs)can be employed to allow simultaneous UL and DL transmissions at the same time–frequency resources as compared to its HD counterpart.It ideally achieves twice the throughput without any additional complexity at user-equipment(UE).This paper covers a detailed survey on FD cellular networks.A series of SIR approaches,UE-UE mitigation techniques are summarized.Various existing MAC protocols and antenna architectures for FD cellular networks are outlined.An overview of security aspects for FD in cellular networks is also presented.Lastly,various open issues and possible research directions are brought up for FD cellular networks.

齿兰环斑病毒RT-qPCR检测方法的建立

【目的】针对齿兰环斑病毒(Odontoglossum ringspot virus,ORSV)建立快速、有效检测方法,对于保障兰花规模化生产有重要意义。【方法】以齿兰环斑病毒为材料,根据NCBI上已登录的齿兰环斑病毒外壳蛋白(coat protein,cp)基因序列分析其序列保守区,使用Primer Premier 5.0设计反转录荧光定量PCR引物及TaqMan荧光探针,建立检测齿兰环斑病毒的一步法RT-qPCR。构建齿兰环斑病毒cp基因克隆质粒,并以该质粒作为标准品进行荧光定量PCR标准曲线测定;以齿兰环斑病毒、建兰花叶病毒(Cymbidium mosaic virus,CymMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)的病毒RNA为模板进行一步法RT-qPCR特异性检测;以10倍倍比稀释的cp基因质粒为模板进行灵敏性检测;用该法对大田18个兰花样品进行齿兰环斑病毒感染情况监测。【结果】齿兰环斑病毒一步法RT-qPCR检测法仅检出齿兰环斑病毒阳性模板,其余模板检测结果为阴性,说明检测方法具有特异性;该方法能够检测到的齿兰环斑病毒cp基因最低拷贝数为130;大田兰花样品感染齿兰环斑病毒的检出率为100%。【结论】一步法RT-qPCR是一种特异性强、灵敏度高,且适合监测实际生产过程中兰花感染齿兰环斑病毒情况的检测方法。

Investigation on influence of alloying on phase transitions of duplex stainless steel based on thermochemical calculation

This paper investigated on influence of different alloying elements added into duplex stainless steel (DSS) on phase transitions using thermochemical methods in comparison with experiment.The results showed that the most possible species in the ferrite phase,austenite phase,σphase,Hcp phase,χphase,and carbide were Cr:Va-type,Fe:Va-type,Ni:Cr:Mo-type,Cr_(2)N-type,Fe_(24)Mo_(10)Cr_(24)-type,and Cr:Mo:C-type,respectively.Furthermore,the Ni,N,Cr,and Mo alloying had significant influences on the transition of each DSS phase.The Ni and N additions obviously raised the temperature at ferrite-1/austenite-1 balance while the Cr and Mo decreased the dual-phase balance temperature.In addition,the Ni addition can promote the precipitating ofσphase at relatively high temperature while the precipitating of Hcp phase at relatively low temperature.The Hcp phase andχphase can be obviously increased by the N addition.The introduction of Cr and Mo notably enhances the precipitation ofσphase.However,the promotion ofχphase precipitation is facilitated by the presence of Mo,while the Cr element acts as an inhibitor forχphase precipitation.Furthermore,the ferrite/austenite ratio tested by experiment was higher than that calculated by thermochemical methods,thus pre-designed solution temperature should be lower about 30-100℃than that calculated by thermochemical methods.

Literature Review of Phase Transformations and Cavitation Erosion of Duplex Stainless Steels

Phase transformation is one of the factors that would significantly influence the ability to resist cavitation erosion of stainless steels. Due to the specific properties of duplex stainless steel, the heat treatment would bring about significant phase transformations. In this paper, we have examined the previous studies on the phase transition of stainless steel, including the literature on the classification of stainless steel, spinodal decomposition, sigma phase transformation, and cavitation erosion of double stainless steel. Through these literature investigations, the destruction of cavitation erosion on duplex stainless steel can be clearly known, and the causes of failure of duplex stainless steel in seawater can be clarified, thus providing a theoretical basis for subsequent scientific research. And the review is about to help assess the possibility of using bulk heat treatment to improve the cavitation erosion (CE) behaviour of the duplex stainless steel 7MoPLUS.

RT-qPCR normalization of reference genes in different lifehistory stages of Gracilaria vermiculophylla(Rhodophyta)

The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.

基于网络药理学和大鼠体内验证的藏药红景天改善脑微循环障碍作用机制研究

目的基于文献研究、网络药理学、分子对接与实验验证的方法,探究藏药红景天改善脑微循环障碍的核心靶点、关键成分和作用机制。方法通过文献和数据库收集红景天化学成分,利用反向药效团匹配预测红景天的潜在靶点;获取脑微循环障碍靶点,并与红景天靶点映射,构建交集靶点蛋白互作网络,获取核心靶点;构建“中药-成分-核心靶点-疾病”调控网络并获取关键成分;进行GO和KEGG富集分析,构建“核心靶点-信号通路-生物过程”网络;进行分子对接验证;采用RT-qPCR和Western blot进行动物实验验证,进一步证实网络药理学分析结果。结果从红景天中筛选出76个活性成分和285个靶点,获取脑微循环障碍靶点1074个,交集靶点97个,核心靶点6个,关键成分6个。分子对接结果表明,有3个关键成分与核心靶点的结合性大于核心靶点蛋白与其原始配体结合性。RT-qPCR结果显示红景天能下调核心靶点CASP3、AKT1 mRNA水平,Western blot检测结果显示藏药红景天能降低CASP3、AKT1蛋白表达(P<0.05)。结论藏药红景天可通过多成分、多靶点、多通路协同改善脑微循环障碍,该研究为临床应用藏药红景天治疗脑微循环障碍提供实验依据。

Deformation Mechanism of Bimodal Structured 2205 Duplex Stainless Steel in Two Yield Stages

A kind of micro/nanostructured 2205 duplex stainless steel(DSS)with uniform distribution of nanocrystals was prepared via aluminothermic reaction method.The analysis of stress-strain curve showed that the fracture strength and elongation of the specimen were 946 MPa and 24.7%,respectively.At present,the research on microstructure of bimodal 2205 DSS at room temperature(RT)mainly depended on scanning electron microscope(SEM)observation after loading experiments.The test result indicates that there are two different yield stages in stress-strain curve of specimen during tensile process.The microstructure of duplex bimodal structured stainless steel consists of two pairs of soft hard regions and phases.By studying deformation mechanism of bimodal structured stainless steel,the interaction between soft phase and hard phase are discussed.The principle of composition design and microstructure control of typical duplex stainless steel is obtained,which provides an important research basis for designing of advanced duplex stainless steel.

热处理对2205双相不锈钢显微组织及性能的影响

采用光学显微镜、显微硬度计和电化学工作站等研究了2205双相不锈钢经不同温度保温不同时间后的显微组织及性能,并分析了其经不同工艺热处理后的γ和α两相比例和析出相的变化。结果表明:2205双相不锈钢初始状态下α和γ相含量分别为41.2%和58.8%。保温时间相同时,随着热处理温度的升高,γ相含量减少,σ相含量先增加后减少,硬度增加,腐蚀电位先降低后增加。保温温度相同时,随着保温时间的延长(0.5~2 h),γ相和σ相含量增加,γ相含量增幅为2.43%~3.87%,σ相含量增幅为0.05%~0.08%,晶粒有长大趋势,硬度逐渐降低,腐蚀电位总体呈降低趋势。当加热温度为950和1000℃时,硬度随保温时间增加而降低的幅度较明显。σ相含量及α/γ比例增加是造成2205双相不锈钢耐腐蚀性能下降的主要原因。