蓝舌病毒核酸检测技术研究进展

蓝舌病毒（BTV）血清型较多,其核酸检测主要涉及通用型检测和分型检测,寻求相应的适宜检测靶基因尤为重要。BTV核酸检测技术是蓝舌病诊断的重要手段,其发展过程主要经历了基因杂交探针技术、RT-PCR检测技术、实时荧光定量PCR检测技术及基因芯片检测技术等;同时,建立和完善高通量BTV筛查技术成为迫切要求。     

罗氏沼虾诺达病毒的核酸检测及其部分序列分析

根据安替列群岛分离的罗氏沼虾诺达病毒株基因组序列(MrNV-ant),制备特异性核酸探针,设计特异性引物,用点杂交和RT-PCR的方法检测在中国境内分离的罗氏沼虾诺达病毒(MrNV-chin).点杂交的方法可以检测出少于26ng的患肌肉白浊症的组织样品中的病毒,或少于25ng的病毒RNA样品;RT-PCR可以检测出少于25pg的RNA样品.扩增的MrNV-chin RNA1序列长858个核苷酸,与MrNV-ant的核苷酸一致率为957%,两者翻译后的氨基酸序列的一致率为99.7%.扩增的MrNV-chin RNA 2序列长1121个核苷酸,与MrNV-ant的核苷酸一致率为92%,两者翻译后的氨基酸序列的一致率为93.2%.因此,MrNV-ant和MrNV-chin应属于同一种病毒的不同分离株.用两株罗氏沼虾诺达病毒的RNA聚合酶序列与其它6株诺达病毒RNA聚合酶序列比较后构建的进化树中,罗氏沼虾诺达病毒与Alphanodavirus的亲缘关系近于与Betanodavirus的亲缘,组成了一个新的分支.     

Characterization of Bluetongue Virus Ribonucleic Acid

An improved purification procedure yielded bluetongue virus free from any single-stranded ribonucleic acid (RNA) component. Double-stranded RNA obtained from purified virus or isolated from infected cells was fractionated into 5 components by means of sucrose gradient sedimentation analysis, and into 10 components by electrophoresis on polyacrylamide gels. The size of these components vary from 0.5 x 10(6) to 2.8 x 10(6) daltons, with a total molecular weight estimate of about 1.5 x 10(7) for the viral nucleic acid. The denaturation of the genome and separation of the resulting fragments are also discussed.     

Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.     

肽核酸在病毒检测与治疗中的应用 *

肽核酸(peptide nucleic acid, PNA)是以多肽骨架取代糖磷酸主链的寡核苷酸类似物,又称第三代反义核酸。PNA的电中性多肽骨架结构,使其保留类似糖磷酸链寡核苷酸高靶标亲和力的同时,比糖磷酸主链具有更强的酶稳定性和热稳定性,已成为当今寡核苷酸类似物研究的热点。一方面,PNA对病毒的复制与突变水平具有的快速、有效和准确的检测性能,对疾病的进一步治疗具有重要意义;另一方面,基于PNA的序列特异性和剂量依赖性,能在基因水平上对病毒的生命周期进行特异性的调控,从而能更有效地实现抑制病毒在宿主细胞中生存和复制的目的。结合近十年来的文献,综述了PNA应用于不同病毒的检测及病毒性疾病治疗的最新进展和作用机制,以期为PNA的临床产品研发提供新的思路。     

肽核酸在病毒检测与治疗中的应用

肽核酸(peptide nucleic acid,PNA)是以多肽骨架取代糖磷酸主链的寡核苷酸类似物,又称第三代反义核酸。PNA的电中性多肽骨架结构,使其保留类似糖磷酸链寡核苷酸高靶标亲和力的同时,比糖磷酸主链具有更强的酶稳定性和热稳定性,已成为当今寡核苷酸类似物研究的热点。一方面,PNA对病毒的复制与突变水平具有的快速、有效和准确的检测性能,对疾病的进一步治疗具有重要意义;另一方面,基于PNA的序列特异性和剂量依赖性,能在基因水平上对病毒的生命周期进行特异性的调控,从而能更有效地实现抑制病毒在宿主细胞中生存和复制的目的。结合近十年来的文献,综述了PNA应用于不同病毒的检测及病毒性疾病治疗的最新进展和作用机制,以期为PNA的临床产品研发提供新的思路。     

A Pair of Novel Primers for Universal Detection of the NS1 Gene from Various Bluetongue Virus Serotypes

Twenty five serotypes of Bluetongue virus （BTV） have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue （BT）. We have therefore developed and evaluated a pair of primers which can detect various serotypes of BTV by RT-PCR. Analysis of the viral protein 7 （VP7） and the non-structural protein （NS1） gene from different serotypes of BTV by DNAstar showed that the 5＇end of the NS1 gene is the most conserved region. The primer pairs （P1 and P2） were designed based on the highly conserved region of NS 1. The novel primers were evaluated by detecting BTV serotypes 1, 3, 5, 8, 10, 11, 21 and 22. The specificity of the primers was estimated by comparing to gene sequences of viruses published in GenBank, and further assessed by detecting BTV serotype 1-12 and Epizootic hemorrhagic disease virus （EHDV） serotype 1-4. The sensitivity and repeatability of PCR with the novel primers were evaluated by successfully detecting the recombinant plasmid pGEM-T121 containing the diagnosed nucleotide sequence. Our results suggest that these unique primers can be used in high throughout and universal detection of the NS1 gene from various BTV serotypes     

HIV-1抗体蛋白印迹确认与核酸检测复核对比研究

应用病毒核酸载量法NASBA和HIV-1 RNA的巢式逆转录PCR(nested RT-PCR)法与HIV抗体蛋白印迹(WB)方法,对经过初筛的44例HIV-1抗体阳性标本进行了对照检测研究。发现了2例(gp160、p24)和1例(gp160g、p120p、66、p24)的特殊阳性样本,经NASBA法和该RT-PCR法核酸检测为阴性;WB确认的4例gp160阳性带、1例p24、p17阳性带和13例p24阳性带,经NASBA法和该RT-PCR法核酸检测也为阴性;而WB确认的其余全部带型的抗体阳性标本经过NASBA法和该RT-PCR法检测均为阳性。该研究表明对只有gp160p、24和gp160、gp120p、66、p24的特殊阳性标本和以p24为主的抗体不确定标本需要用RT-PCR或NASBA方法进行核酸检测,以进一步确认。     

RT—PCR检测蓝舌病毒技术的建立

蓝舌病毒 (ＢｌｕｅｔｏｎｇｕｅＶｉｒｕｓ,ＢＴＶ)是呼肠孤病毒科 (Ｒｅｏｖｉｒｉｄａｅ)环状病毒属 (Ｏｒｂｉｖｉｒｕｓ)的代表种 ,含10个节段的双链ＲＮＡ(ｄｓＲＮＡ)作基因组。其中 ,Ｌ2节段编码ＢＴＶ型特异性抗原ＶＰ2 ,Ｌ3节段和Ｓ7节段编码群特异性抗原多肽ＶＰ3和ＶＰ7。其中 ,ＶＰ7是ＢＴＶ粒子的主要结构多肽之一 ,其编码基因序列保守。ＶＰ7具有高度的抗原性 ,能刺激被感机体产生强的群特异性免疫反应[1] 。蓝舌病毒的易感宿主是牛、羊及野生反刍动物 ,死亡率高达 6 0 %～ 70 %以上 ,并且至今仍无有效的防治措施 ,对畜牧业…     

RT-PCR检测蓝舌病毒技术的建立

蓝舌病毒(Bluetongue Virus,BTV)是呼肠孤病毒科(Reoviridae)环状病毒属(Orbivirus)的代表种,含10个节段的双链RNA(dsRNA)作基因组.其中,L2节段编码BTV型特异性抗原VP2,L3节段和S7节段编码群特异性抗原多肽VP3和VP7.其中,VP7是BTV粒子的主要结构多肽之一,其编码基因序列保守.VP7具有高度的抗原性,能刺激被感机体产生强的群特异性免疫反应[1].     

蓝舌病病毒Z1株VP7基因的克隆及序列分析

用ＲＴ－ＰＣＲ扩增我国蓝舌病毒Ｚ１株的ＶＰ７基因,直接将其克隆至ｐＧＥＭ－Ｔ载体中,用限制性内切酶ＥｃｏＲＩ分析经蓝／白斑筛选和ＰＣＲ鉴定的重组质粒,用ＤＩＧ标记克隆片段制成探针与病毒基因组进行Ｎｏｒｔｈｅｒｎ ｂｌｏｔ杂交,证明插入片段为ＢＴＶ ＶＰ７基因特异性片段。采用Ｓａｎｇｅｒ双脱氧终止法测定ｃＤＮＡ片段的核苷酸序列,将这一序列与国外已发表的９株蓝舌病毒及相关的环状病毒的ＶＰ７基因进行了比     

核酸检测技术进展及其在SARS-CoV-2核酸检测中的应用

2020年初至今,新型冠状病毒肺炎(COVID-19)疫情仍在全球多个国家流行,给全球公共卫生安全造成了严重威胁.中国在应对COVID-19的实践中,最先完成病毒核酸测序并共享序列信息,最早有效控制疫情蔓延、恢复生产,并在病毒作用人体机制研究、疫苗研发、中和性抗体发现等环节均处于世界前沿.其中,针对病毒核酸的检测工作—...     

Characterization of Nucleic Acid of Pichinde Virus

The nucleic acid of Pichinde virus was found to be single-stranded ribonucleic acid (RNA) as determined by sensitivity to ribonuclease, by alkaline degradation, by buoyant density in cesium sulfate, and by analysis of the base composition. The RNA of the virion could be separated into five components which had sedimentation coefficients corresponding to 31S, 28S, 22S, 18S and 4 to 6S. The 28S, 18S, and possibly the 4 to 6S RNAs appear to be derived from host cell components incorporated into the virion, whereas the 31S and 22S components appear to represent the genome of the virus.     

基于CRISPR/Cas系统的核酸生物传感器

近年来,CRISPR/Cas系统已经成为转录调控和基因组编辑的重要工具。除了在基因编辑领域的贡献,CRISPR/Cas系统独特的靶核酸顺式切割和非特异性单链核酸反式切割能力,在开发核酸检测的新型生物传感器方面展现出巨大潜力。构建基于CRISPR/Cas系统高灵敏度生物传感器的关键通常依赖其与不同信号扩增策略,诸如核酸扩增技术或特定信号转导方法的结合。基于此,本文旨在通过介绍不同类型的CRISPR/Cas系统,全面概述基于该系统的核酸检测生物传感器的研究进展,并重点对结合核酸扩增技术（PCR、LAMP、RCA、RPA和EXPAR）、灵敏的信号转导方法（电化学和表面增强拉曼光谱）和特殊结构设计生物传感的三大类型信号放大策略的CRISPR/Cas生物传感器进行总结和评论。最后,本文对目前的挑战以及未来的前景进行展望。     

南方水稻黑条矮缩病毒快速检测

南方水稻黑条矮缩病毒(southern rice black-streaked dwarf virus,SRBSDV)引起的新水稻矮缩病近年在越南北部和我国南方各省爆发,准确快速地检测病毒是病害预测的关键。本文针对该病毒的S10序列设计了一对新的引物S10F/S10R,利用一步法RT-PCR,对2010年5~8月间湖南省下属各县送来的疑似感染该病毒引起的水稻矮缩病样本进行了检测,结果显示,新引物能有效地区分阳性样品和非阳性样品。同时对PCR产物进行了序列测定,结果表明序列均与已发表的南方水稻黑条矮缩病毒序列(登录号:EU523360和EU784840)达约99%的同源性。还在此基础上构建了系统发育树,发现SRBSDV湖南、广东和海南分离物病毒位于一个相对独立的分支。研究发现相对以往的巢式RT-PCR,本文采用的新引物及一步法RT-PCR能快速检测南方水稻黑条矮缩病毒,简化了操作步骤,缩短了检测时间,适合在SRBSDV检测和病害预测中应用。     

核酸试纸条检测方法研究进展*

凝胶电泳、实时荧光PCR等常规核酸检测方法存在操作繁琐、设备昂贵、反应时间长等局限性。随着核酸检测市场规模的大幅提升,常规检测方法已无法满足临床诊断、检验检疫的需求。核酸试纸条(nucleic acid detection strip,NADS)是一种新兴的核酸检测方法,具有灵敏度高、操作便捷、结果可视化、成本低且耗时短等优势,在基础研究与临床诊断等领域受到广泛关注。综述近年来NADS的检测方法及研究进展,系统总结该技术的原理、应用及临床潜在转化价值,以期为NADS的进一步开发、利用提供借鉴。     

核酸纯化柱提取核酸定量检测丙型肝炎病毒RNA的临床应用

目的:探究核酸纯化柱提取核酸定量检测血浆标本中丙型肝炎病毒核糖核酸(HCV-RNA)的临床应用效果。方法:将2013年8-11月期间我院600例抗-HCV阳性丙肝患者的样本,按随机字数表法分为研究组对照组各300例,分别采用核酸纯化柱和酚-氯仿提取法检测,采用实时荧光定量聚合酶链反应(FQ-PCR)技术定量检测两组HCV-RNA水平。结果:研究组检测阳性率为87.67%(263/300),显著高于和对照组的54.0%(162/300),差异有统计学意义(X2=82.296,P=0.000);两组HCV-RNA检测水平差异无统计学意义(u=1.721,P=0.067)。结论:核酸柱提法定量检测血浆HCV-RNA操作简单、快速,分离效率高,容易掌握,值得临床推广。     

Universal Labeling Chemistry for Nucleic Acid Detection on DNA-Arrays

Abstract

We show here a new and efficient aqueous chemistry for labeling of any class of nucleic acids for their detection on DNA chip. The labels contain a diazo function as reactive moiety and biotin as detectable unit. The highly selective reaction of diazo group on the phosphate does not disrupt base pairing recognition and hybridization specificity.     

Reticuloendotheliosis Virus Nucleic Acid Sequences in Cellular DNA

Reticuloendotheliosis virus 60S RNA labeled with (125)I, or reticuloendotheliosis virus complementary DNA labeled with (3)H, were hybridized to DNAs from infected chicken and pheasant cells. Most of the sequences of the viral RNA were found in the infected cell DNAs. The reticuloendotheliosis viruses, therefore, replicate through a DNA intermediate. The same labeled nucleic acids were hybridized to DNA of uninfected chicken, pheasant, quail, turkey, and duck. About 10% of the sequences of reticuloendotheliosis virus RNA were present in the DNA of uninfected chicken, pheasant, quail, and turkey. None were detected in DNA of duck. The specificity of the hybridization was shown by competition between unlabeled and (125)I-labeled viral RNAs and by determination of melting temperatures. In contrast, (125)I-labeled RNA of Rous-associated virus-O, an avian leukosis-sarcoma virus, hybridized 55% to DNA of uninfected chicken, 20% to DNA of uninfected pheasant, 15% to DNA of uninfected quail, 10% to DNA of uninfected turkey, and less than 1% to DNA of uninfected duck.