盐地碱蓬SsDHN基因的功能分析

脱水素(DHN)是一种在逆境下起重要作用的功能性蛋白.为了分析脱水素蛋白在抗盐碱方面的作用机制,本研究以从盐地碱蓬中克隆获得的脱水素(SsDHN)基因为研究对象,对基因序列进行生物信息学分析,结果表明盐地碱蓬的脱水素蛋白为部分无序的亲水性蛋白,含1个S-片段和3个K-片段,属于SKn型脱水素蛋白.通过实时荧光定量PCR...     

猪附红细胞体荧光定量PCR检测方法的建立及应用

摘要: 根据GenBank已登录的猪附红细胞体（M.suis）推测的功能性蛋白基因ORF2序列设计引物和TaqMan荧光探针,以定量的10倍系列稀释含M.suis部分ORF2基因的T载体重组质粒（pGEX-T/M.suis）为标准品,进行荧光定量PCR扩增并制作了标准曲线,经对荧光定量PCR的反应条件进行优化,建立了M.suis的TaqMan荧光定量PCR检测方法（Taqman FQ-PCR）;对所建立的FQ-PCR检测方法进行了敏感性、特异性和重复性实验,并对疑似M.suis感染临床抗凝全血样品进行了检测应用。结果显示：标准曲线的曲线循环阈值与模板浓度有良好的线性关系,相关系数为0.998;所建立的FQ-PCR方法检测灵敏度可达10个拷贝/μL,比对照常规PCR灵敏度高100倍;FQ-PCR方法特异性高,对pGEX-T/M.suis重组质粒扩增呈现阳性反应曲线,而对8个对照细菌、病毒和寄生虫DNA扩增曲线均呈现阴性反应;对不同浓度的pGEX-T/M.suis重组质粒分别重复扩增2次,重复结果良好;用该方法对24份临床疑似M.suis感染样品进行了应用检测,结果有20份样品为阳性,阳性检出率高于常规PCR方法。     

Development of real-time PCR assays for the detection and differentiation of Australian and European ranaviruses

Serious systemic disease in fish and amphibians is associated with the ranaviruses, epizootic haematopoietic necrosis virus (EHNV) and Bohle iridovirus (BIV) in Australia, and European sheatfish virus (ESV) and European catfish virus (ECV) in Europe. EHNV, ESV and ECV are recognized causative agents of the OIE (Office International des Epizooties) notifiable systemic necrotizing iridovirus syndrome and are currently identified by protein-based assays, none of which are able to rapidly identify the specific agents. The aim of this study was to develop TaqMan real-time PCR assays that differentiated these viruses using nucleotide sequence variation in two ranavirus genes. A conserved probe representing 100% sequence homology was used as a reference for virus-specific probes. The virus-specific probes produced a similar signal level to the conserved probe while those probes binding to non-target viral DNA produced an altered fluorescent curve. The pattern of probe binding was characteristic for each virus. Sensitivity, specificity and dynamic range of the assay were assessed. The test is currently useful as a research and initial screening tool, with the potential to become a sensitive and specific method for detection and differentiation of ranaviruses with further development.     

Molecular diagnosis and direct quantification of cereal cyst nematode (Heterodera filipjevi) from field soil using TaqMan real-time PCR

Heterodera filipjevi continues to be a major threat to wheat production worldwide. Rapid detection and quantification of cyst nematodes are essential for more effective control against this nematode disease. In the present study, a TaqMan-minor groove binder (TaqMan-MGB) probe-based fluorescence quantitative real-time PCR (qPCR) was successfully developed and used for quantifying H. filipjevi from DNA extracts of soil. The primers and probe designed from the obtained RAPD-SCAR marker fragments of H. filipjevi showed high specificity to H. filipjevi using DNA from isolates-confirmed species of 23 Heterodera spp., 1 Globodera spp. and 3 Pratylenchus spp. The qPCR assay is highly sensitive and provides improved H. filipjevi detection sensitivity of as low as 4^–3 single second-stage juvenile (J2) DNAs, 10^–3 female DNAs, and 0.01 μg μL^–1 genomic DNAs. A standard curve relating to the threshold cycle and log values of nematode numbers was generated and validated from artificially infested soils and was used to quantify H. filipjevi in naturally infested field soils. There was a high correlation between the H. filipjevi numbers estimated from 32 naturally infested field soils by both conventional methods and the numbers quantified using the qPCR assay. qPCR potentially provides a useful platform for the efficient detection and quantification of H. filipjevi directly from field soils and to quantify this species directly from DNA extracts of field soils.     

五指毛桃转录组及黄酮类化合物生物合成关键基因分析

五指毛桃含有香豆素类、黄酮类等化学成分,具有抗炎、抑菌、抗病毒和肿瘤的药用价值.但是,其转录组和功能基因组等分子水平的研究较少报道.本研究首次采用RNA-seq高通量测序技术对五指毛桃的根、茎和叶进行转录组测序,共获得104335条unigenes,平均长度为578 bp,有62142条unigenes被注释到Nr、G...     

五倍子蜂蜜TaqMan探针实时荧光定量PCR检测方法的建立

为五倍子蜂蜜的鉴定检测提供技术支撑,利用TaqMan探针实时荧光定量PCR技术,以五倍子树基因组中ITS基因为靶序列,设计并筛选出特异性引物探针,通过特异性、灵敏度和重复性等试验,建立五倍子蜂蜜TaqMan探针实时荧光PCR检测方法。结果表明:建立的方法特异性良好,生成的标准曲线有良好的线性关系,相关系数(R2)为0.998,最低检测限为0.2pg DNA/反应。该方法特异性强、灵敏度高、结果可靠。     

Rapid identification of eels <Emphasis Type="Italic">Anguilla japonica</Emphasis> and <Emphasis Type="Italic">Anguilla anguilla</Emphasis> by polymerase chain reaction with single nucleotide polymorphism-based specific probes

ABSTRACT:   Standard molecular techniques, such as sequencing and restriction fragment length polymorphism analysis after polymerase chain reaction (PCR) amplification are relatively complicated, and species identification can take a long time when using such techniques. We established a quick method, using PCR with species-specific TaqMan Minor Groove Binder (MGB) probes based on single nucleotide polymorphism (SNP) to distinguish the two eel species Anguilla japonica and Anguilla anguilla . This method can be used in processed products. Partial sequences of the mitochondrial 16S rRNA gene were compared between A. japonica and A. anguilla to design a primer pair common to both A. japonica and A. anguilla and probes specific to A. japonica and A. anguilla . Different fluorescence intensities were produced in two PCR microtubes each containing A. japonica - and A. anguilla -specific probes for one target sample. We observed the fluorescence intensity of PCR products in microtubes under ultraviolet transillumination, with similar results to those obtained by real-time PCR. Therefore, SNP-based PCR is a powerful tool for identifying materials of processed foods from either A. japonica or A. anguilla .     

蓝舌病病毒、流行性出血病病毒和帕利亚姆血清群病毒三重RT-qPCR检测方法的建立及应用

目的 建立蓝舌病病毒(Bluetongue virus,BTV)、流行性出血病病毒(Epizootic hemorrhagic disease virus,EHDV)及帕利亚姆血清群病毒(Palyam serogroup virus,PALV)快速筛查和诊断的三重RT-qPCR检测技术。方法 选择BTV的NS3、EHDV的NS1以及PALV的VP7基因作为靶基因,设计3组特异性引物和探针,建立3种病毒的三重RT-qPCR检测方法。对检测方法的灵敏度、特异性和重复性进行验证,同时使用我国分离的BTV、EHDV与PALV不同血清型毒株和对应的阳性血液样本,与24个BTV血清型及6个EHDV血清型参考毒株对该三重法的检测效果进行验证。结果 三重RT-qPCR的扩增效率均可达90%以上,对3种病毒核酸拷贝数的检测下限均为10 μL^-1级别,与单重法的检测灵敏度相当;与阿卡斑病毒、小反刍兽疫病毒、口蹄疫病毒和牛流行热病毒之间无交叉反应;Ct值批内、批间变异系数均在2%以内;可检测出24种BTV血清型、6种EHDV血清型与3种PALV血清型,具有群特异性;可有效检测血液中的病毒核酸,检测结果与单重法一致。结论 本研究建立的BTV、EHDV与PALV三重RT-qPCR具有良好的敏感性、特异性和重复性,可用于临床样本中对3种病毒的同时诊断与筛查。     

柑橘品种对不同基因型柑橘衰退病毒的抑制及变异的影响

为研究不同柑橘品种对柑橘衰退病毒（Citrus tristeza virus,CTV）不同基因型的抑制效果,将T36、T30、VT和T3等基因型CTV的毒源分别接种于‘赛蒙斯’甜橙、‘邓肯’葡萄柚、‘墨西哥莱檬’和‘强徳勒柚’,运用RT-qPCR技术检测被接种植株中CTV复制水平的差异。结果表明,‘墨西哥莱檬’最适于CTV各基因型的复制。‘邓肯’葡萄柚中VT、T3和T36基因型的复制水平最低。4种柑橘品种对T36基因型CTV都有明显的抑制作用。通过对CTV的CP基因进行分析发现,VT基因型CTV在‘赛蒙斯’甜橙中发生了较大变异。     

TaCBL基因在小麦与叶锈菌互作过程中的表达分析

利用实时定量PCR技术对小麦与叶锈菌互作过程中的钙调磷酸酶B类似蛋白相关基因的表达进行了检测分析。结果表明,在亲和组合接种后的16h,TaCBL1、TaCBL3、TaCBL4、TaCBL7基因的相对表达量明显增加,分别达到了对照的近8,2,4.5,14倍的水平,而在不亲和组合中,这4个基因在不同时间点的表达水平与对照相近;TaCBL2和TaCBL9的表达趋势在亲和组合和不亲和组合间大致相似,但在不亲和组合中其表达的峰值出现的时间要早于亲和组合;TaCBL6在2个组合的表达变化基本一致。以上结果初步表明,TaCBL可能参与了小麦抵抗叶锈菌侵染的基础防卫反应,其中TaCBL2和TaCBL9在抵抗叶锈菌侵染过程中发挥正调控作用,而TaCBL1、TaCBL3、TaCBL4和TaCBL7的高表达削弱了寄主的抗病性从而更利于叶锈菌的侵染。为进一步深入研究Ca2+信号通路中钙调磷酸酶B类基因在小麦与叶锈菌互作过程中的作用机制奠定了试验基础。