Texel和乌珠穆沁绵羊妊娠中后期胎儿背最长肌中 AKT基因的发育表达模式

选择绵羊胎儿期骨骼肌中表达稳定性高的内参基因,探讨AKT基因在绵羊妊娠中后期背最长肌中的发育性表达模式。以Texel和乌珠穆沁绵羊为研究对象,采用荧光定量PCR技术检测SDHC、YWHAB、RPLPO、RPS18、B2M 5个内参基因的mRNA表达水平,同时检测AKT基因mRNA丰度在其胎儿期70、85、100、120和135 d间的表达变化,并分析其在品种间和不同生长阶段间的表达差异及其对肌肉生长发育的影响。5个内参基因的定量结果经GeNorm程序统计学分析处理,表达稳定性由高到低排序为RPS18＞RPLPO＞SDHC＞B2M＞YWHAB,确定用RPLPO、RPS18和SDHC 3个基因校正绵羊胎儿期不同生长阶段的目的基因表达量。在5个发育阶段中,两个品种绵羊胎儿AKT基因表达模式有一定差异,AKT表达量在Texel绵羊85和120 d时均有明显的增加,而在乌珠穆沁绵羊的100 d时出现1个表达峰,这与试验对两品种绵羊组织学分析的结果是一致的,进一步印证了Texel绵羊在85和120 d以及乌珠穆沁绵羊在100 d时存在肌发生波的推测。以上结果初步揭示绵羊妊娠中后期生长发育过程中AKT基因表达的发育性变化模式和品种差异,为深入研究AKT基因的作用机制提供了理论基础。     

应用实时荧光定量RT-PCR高效检测葡萄病毒B

 本研究建立了葡萄病毒B的 SYBR GreenⅠ实时荧光定量RT-PCR(RT-qPCR)检测技术。该技术标准曲线扩增效率102.4%,相关系数0.999,最低检测限达10^-4倍稀释cDNA,灵敏度为常规RT-PCR的100倍。重复性试验组内和组间变异系数分别为0.00%~0.65%和0.02%~2.00%,表明检测稳定性好。该技术对田间葡萄样品检测适用范围广,对枝条和老叶柄检测效果最好,冬季枝条和春夏秋季所有老叶柄样品检出率均为100%,与常规RT-PCR检测结果一致。对于其他季节或部位样品,RT-qPCR检出率(43% ~74%)则普遍高于常规RT-PCR(5% ~71%),特别是春季样品和春夏秋季所有嫩叶样品,检出率比常规RT-PCR分别高31% 和38%。对来自我国13个省21个品种的52份田间葡萄样品检测结果表明, RT-qPCR共检测到6个样品为阳性,检出率11.5%,为常规RT-PCR(检出率5.8%)的2倍。     

双重RT-PCR法同步检测单头异沙叶蝉携带的两种小麦病毒

异沙叶蝉可传播小麦矮缩病毒Wheat dwarf virus(WDV)和小麦黄条纹病毒Wheat yellow striate virus(WYSV)两种病毒。本文根据WDV和WYSV的基因序列分别设计两种病毒的特异性引物对,以含有上述两种病毒的异沙叶蝉样品总RNA为模板,以随机引物为3′端通用引物反转录获得cDNA,然后在同一个PCR反应体系加入两对引物,分别得到与预期相符的773 bp和322 bp扩增产物条带。通过对引物浓度、dNTP和rTaq用量以及退火温度等条件进行优化,建立了能在同一异沙叶蝉体内检测WDV与WYSV两种小麦病毒的双重RT-PCR方法。该双重PCR方法特异性强、敏感性高,可以快速准确地在一个体系里同步检测介体昆虫体内的两种病毒,有效地检测虫体内病毒带毒率,这些结果可为病毒预测预报和病害防治提供参考。     

双重RT-PCR同时快速检测H5亚型禽流感病毒和新城疫病毒强毒株

本研究针对AIV H5的血凝素(HA)基因和vNDV的融合蛋白(F)基因设计两对引物,建立了单管同时检测AIVH5和vNDV的双重RT-PCR(dRT-PCR),该方法能在单一反应管内同时检测AIVH5和vNDV,不与其它亚型AIV、弱毒NDV毒株以及其它病原体发生非特异性扩增;对含有AIV H5 HA基因和vNDV F基因重组质粒DNA的最低检测限分别为20.6和406fg,敏感性与单项RT-PCR相同;从样本处理到报告结果仅需5h。对24份临床疑似样本进行检测,AIVH5均为阴性,vNDV阳性18份,PCR产物测序证明为靶基因序列,其具有vNDV F基因的特征性序列,随机选9份vNDV阳性样本接种SPF鸡胚,分离出6株vNDV,病毒分离率为6/9;对100 ELD50的AIV H5N1和100 ELD50的vNDV人工同时感染5日龄SPF鸡的脑、肝脏、肺脏和泄殖腔棉拭子进行dRT-PCR检测,AIV H5的检测率为4/5,3/5,5/5,4/5,vNDV的检测率为3/5,5/5,4/5,3/5,而对H9N2和LaSota毒株实验同时感染样本及阴性对照样本的检测均为阴性。可见本研究建立的dRT-PCR为AIV H5和vNDV诊断、监测、检疫及分子流行病学调查提供了特异性强、操作简单、检测速度快、成本低廉的新方法。     

库尔勒香梨主要病毒多重RT-PCR检测技术研究

 利用nad5基因作为内标系统,研究建立了库尔勒香梨上苹果茎痘病毒(ASPV)、苹果褪绿叶斑病毒(ACLSV)和苹果茎沟病毒(ASGV)等的RT-PCR检测技术,在此基础上研究建立了库尔勒香梨多重RT-PCR内标检测系统。并对回收的特异片段进行了克隆、鉴定和测序。测序结果表明:库尔勒香梨上的ACLSV与GenBank中D14996序列(日本苹果上的ACLSV分离物)中的6860~7536bp片段有116个碱基的差异,序列相似性为83.75%;库尔勒香梨上的ASPV与GenBank中D21828序列(德国梨脉黄病毒相关分离物)中的8869~9238bp片段有72个碱基的差异,序列相似性为79.5%;库尔勒香梨上的ASGV与GenBank中AB004063序列(日本ASGV分离物)中的6039~6311bp片段有21个碱基的差异,序列相似性为92.3%。     

我国玉米上一个水稻黑条矮缩病毒重排体的ＯＲＦ序列(英文)

 玉米粗缩病20世纪50年代和90年代中后期在我国部分地区严重发生,2008年至今, 该病在黄淮海地区又呈暴发趋势^[1]。引起我国北方玉米粗缩病的主要是水稻黑条矮缩病毒(Rice black-streaked dwarf virus,RBSDV)^[2]。田间寄主植物和传毒昆虫灰飞虱(Laodelphax striatellus Fallen)的发生数量、带毒率与玉米粗缩病的发生密切相关^[1]。因此,建立快速灵敏的RBSDV检测体系,明确RBSDV的田间寄主,对有效控制玉米粗缩病具有重要意义。     

Genome-based PCR Primers for Specific and Sensitive Detection and Quantification of Xylella fastidiosa

Xylella fastidiosa is an important pathogen of many commercial crops. Detection of X. fastidiosa is difficult due to low concentrations of the bacteria in insects and asymptomatic plant tissue, and non-uniform distribution in infected plants. A dual purpose conventional PCR and quantitative PCR (TaqMan™) system was developed for the generic detection of X. fastidiosa strains. Primers HL5 and HL6, designed to amplify a unique region common to the sequenced genomes of four Xylella strains, amplified a 221 bp fragment from strains associated with Pierce’s disease of grapes, almond leaf scorch, and oleander leaf scorch disease and from DNA from an Xf strain associated with citrus variegated chlorosis. Standard curves were obtained using concentrations of Xylella ranging from 5 to 10⁵ cells per reaction in water and grape extracts and 10–10⁵ cells in insect DNA. Regression curves were similar, with correlation coefficients of r ² > 0.97. In quantitative PCR, C_t values ranged between 20 and 36 cycles for 5–10⁵ bacterial cells per reaction. No amplicons were obtained with several non-Xf bacterial strains tested including related plant pathogenic, grape endophytic bacteria and endosymbiotic bacteria isolated from glassy-winged sharpshooters. The method was evaluated for clinical diagnosis of Xf in grapes, almonds and insect vectors. The procedure described is reliable for detection of the pathogen with a high degree of sensitivity and specificity.     

Improved method for assaying maize plant resistance to maize rough dwarf disease by artificial inoculation and real-time RT-PCR

The study of maize rough dwarf disease (MRDD) and breeding for resistance requires inoculation of maize plants by means of planthoppers. The plant age, insect density and inoculation duration are main factors in the success of maize rough dwarf disease inoculation. These parameters were tested using a susceptible maize inbred line Ye478. Using one or two-leaf plants, 15 planthoppers per plant and a five day inoculation duration, the line Ye478 was the most susceptible with 100% diseased plants; F112132 was moderately susceptible with 60% diseased plants and 90110 and F022411 were resistant without any disease. The results were consistent with those from six years of field studies. Using enzyme-linked immunosorbent assay (ELISA) and real-time quantitative RT-PCR, rice black-streaked dwarf virus was detected in severely diseased plants. The plants were rated from 0 to 3 according to their symptoms at the time of flowering. Plants scoring 0, 1 and 2 could not be distinguished by ELISA, only by real-time quantitative RT-PCR. All of the plants with a score of 3 were positive by ELISA and real-time quantitative RT-PCR. The significant differences in the average viral contents in plants with different symptom ratings could be distinguished by using real-time RT-PCR.     

应用TaqMan MGB探针技术检测菜豆荚斑驳病毒

根据菜豆荚宽驳病毒(Beanpod mottle virus,BPMV)不同分离株外壳蛋白基因(coat protein,CP)的保守序列,设计了特异性引物与TaqMan MGB荧光探针,建立了BPMV的实时荧光RT-PCR检测方法.该方法与酶联免疫检测方法相比,灵敏度提高了100倍,具有快速、灵敏和高特异性的优点,适于对BPMV的快速检测.应用该方法,对来自美国不同地区的携带有BPMV的大豆样品进行检测,均能够得到BPMV的典型扩增曲线,表明引物与探针对BPMV不同分离株具有良好的简并性.     

尤力克柠檬上一种新病害的生物学特性及RT-PCR检测

近年我国云南瑞丽市的一果园尤里克柠檬上出现一种引起叶片皱缩、反卷或呈船形,叶背水浸状、侧脉脉明、黄化的柑橘新病害。为明确该病害的病原及其生物学特性,通过田间观察、病样接种、电镜观察,并基于柑橘黄脉病毒(Citrus yellow vein clearing virus,CYVCV)外壳蛋白基因设计引物对病样进行RT-PCR检测。结果表明,该病是一种嫁接传染性病害,能侵染柠檬和酸橙品种且症状明显,并能摩擦接种到辣椒和豇豆等草本植物。该病发病最适温度为18~24℃。春梢嫩叶症状表现明显,夏梢和秋梢基本不表现症状,而晚秋梢也表现出较春梢稍弱的典型症状。引致该病的病毒为(13~15)nm×(400~1 000)nm大小的联合丝状病毒。RT-PCR扩增产物为614 bp,且其与CYVCV外壳蛋白序列相似性达97%以上,表明该病害是由CYVCV引起的柑橘黄脉病,并初步建立了该病的RT-PCR检测技术。