PCR技术在猴免疫缺陷病毒(SIV)感染模型中的应用

目的(1)建立RT PCR方法,定性测定SIV感染猴血浆中病毒RNA,比较其与传统血浆病毒分离方法的敏感性;(2)建立DNA PCR方法,检测SIV感染猴外周血淋巴细胞(PBMCs)中的前病毒DNA。(3)检验DNA PCR和RNA PCR方法在猴SAIDS模型应用中的实用性和可操作性。方法用SIVmac251静脉感染恒河猴,定期采血,从血浆中提取病毒RNA,以RNA为模板通过RT PCR法扩增,凝胶电泳定性;从感染猴PBMC中提取带有整合的SIV前病毒DNA的细胞基因组DNA,巢式PCR扩增,凝胶电泳定性。结果DNA PCR和RNA PCR经两轮扩增后均得到一长度为477bp的特异条带,测序鉴定确为目的片段。9只实验猴感染SIV后7d,RNA PCR结果为79阳性,DNA PCR结果为100%阳性,而血浆病毒分离只有59阳性;此后一直到感染后的42d,RNA PCR和DNA PCR的结果一直为100%阳性,而血浆病毒分离阳性率在感染后35d下降到49,到42d时下降为零。结论PCR方法比病毒分离方法的敏感性高。尤其是DNA PCR,既可检测具有活跃病毒复制的受感染细胞,又可检测那些携带病毒处于转录休眠期的细胞,所以在感染的早期和中后期———血浆病毒水平较低的情况下或病毒处于潜伏感染的阶段,它作为猴艾滋病(SAIDS)模型病毒学指标之一有其必要性和重要性。这个指标的检测方法应该是较血浆病毒RNA检测更为敏感。     

犬细小病毒的PCR诊断试剂盒的研制

目的　建立检测犬细小病毒的PCR诊断试剂盒。方法　通过在犬细小病毒 (CPV)的基因组中设计的特异性寡核苷酸引物 ,利用PCR技术研制犬细小病毒的PCR诊断试剂盒。结果　在特定的反应条件下 ,使用该试剂盒能特异地扩增含有犬细小病毒的样品 ,且能检出痕量 (10ng)的犬细小病毒DNA ,被测粪样只需进行简单煮沸就能进行PCR反应 ,该试剂盒能在常温下保存 1周以上、4℃保存 1年以上。同血凝试验 (HA〕及单克隆抗体ELISA方法比较 ,对 6 6份样品进行检测 ,显示该PCR试剂盒具有特异、灵敏等优点。结论　成功研制了犬细小病毒的PCR诊断试剂盒 ,利用该试剂盒能从DNA水平上对犬细小病毒进行监控     

不同DNA提取法对腺病毒和细小病毒PCR检测效果评估

分别利用硫氰酸胍(Guanidine thiocyanate,GuScN)抽提法、螯合树脂处理法和蛋白酶K-酚/氯仿抽提法从粪便样品中制备腺病毒DNA(dsDNA)或细小病毒DNA(ssDNA)然后进行PCR检测。结果显示硫氰酸胍抽提法、螯合树脂处理法能有效地去除粪便中影响PCR扩增的抑制物,提高PCR检测的敏感性,而传统蛋白酶K-酚/氯仿抽提法不能有效地去除PCR扩增的抑制物,影响PCR检测结果。在检测粪便中腺病毒时,硫氰酸胍、螯合树脂和蛋白酶-酚/氯仿抽提法分别允许检测5TCID_{50相似文献}   

猴 B 病毒血清学和 PCR 检测结果比较

目的：比较猴B病毒血清抗体和病毒PCR检测结果,阐明动物感染后病毒在机体内的存在状况。方法：采集成年猴血清和三叉神经组织,首先通过ELISA方法检测血清中B病毒抗体,然后采用B病毒舡和徊基因引物通过PCR方法扩增血清DNA和三叉神经组织DNA,比较2种方法的检测结果,并对扩增产物进行序列分析。结果：22份猴血清中,B病毒抗体呈阳性的有13份（59．1％）;PCR结果显示,抗体阴性动物及所有血清DNA模板中均无阳性扩增,但在13份抗体阳性动物的三叉神经组织DNA样品中,PCR阳性4份（30．8％）;gL和gD基因扩增条件及产物分析表明,舡基因的GC含量为64．1％,gD为74．2％,且舡的扩增条件和效果明显优于gD。结论：B病毒感染猴后,将在部分动物神经节中建立潜伏,而础基因更适合作为分子鉴定的靶标。     

开发使用DNA探针结合粒子的检体DNA浓缩法

日本合成橡胶公司和日本罗氏公司小组使用结合了DNA探针的胶乳粒子,开发了简便且高灵敏地提取、浓缩检体中的病原菌和病毒DNA的技术。与神奈川县卫生研究所病毒部部长今井光信等合作使用这项技术用AIDS患者的血液进行检测实验。结果表明,比用普通聚合酶链反应法(PCR)的检测灵敏度高数十倍。 PCR反应是用基因扩增法扩增检测对象——基因高效率地检测的方法。但是,PCR反应的试剂量限于数十微升,所以其界限为数十微升。一旦不含检测对象就检测不出来。如果使用日本合成橡胶公司和日本罗氏开发的技术,在PCR前就能浓缩标记DNA(约330倍),据说在1ml血液中有1个分子,使用PCR     

基于PCR方法的美国白蛾核型多角体病毒早期检测

根据美国白蛾核型多角体病毒(Hyphantria cunea nucleopolyhedrovirus, HcNPV) pe38基因设计两对引物, 利用PCR法检测HcNPV基因组DNA, 分别扩增出长为994和614 bp的片段, 经测序确定为HcNPV pe38基因片段。应用两对引物分别检测了美国白蛾病虫的总DNA和获得的病毒多角体, 其检测最低量分别为1 fg病虫总DNA和3~4 OBs/mL。用不同浓度的病毒感染试虫后, 不同时间取其血淋巴用PCR方法检测。结果表明, 接毒量为3.53×109 OBs/mL时, 24 h后可检出病毒DNA;接毒量为3~4 OBs/mL时, 120 h后可检出。病毒可检出时间随接毒浓度的递减而延后。     

中国番茄黄化曲叶病毒DNAβ缺失突变体与异源病毒的互作研究

与一些单组份双生病毒伴随的卫星DNAβ是辅助病毒在自然寄主上引起典型症状所必需的致病相关分子。迄今发现的所有DNAβ分子在互补链上都含有一个位置和大小保守的βC1基因。对中国番茄黄化曲叶病毒的缺失βC1基因的DNAβ突变体(DNAΔC1β)与异源病毒接种后的致病性及稳定性进行了研究。PCR和Southern杂交证实该突变体能利用赛葵黄脉病毒、烟草曲茎病毒、中国胜红蓟黄脉病毒、假马鞭曲叶病毒等多种异源双生病毒复制并系统侵染本氏烟。接种后65 d内的PCR检测和序列分析显示,该突变体与其它辅助双生病毒接种后在寄主细胞中的复制是稳定的。     

猕猴巨细胞病毒(RhCMV)nested PCR检测方法的建立与初步应用

目的建立猴巨细胞(RhCMV)病毒的nested PCR检测方法,并初步应用。方法根据GenBank中报道的RhCMV全基因序列,针对其中的保守区域Rh85设计两对引物进行nested PCR反应,利用此方法对20份猕猴全血标本进行检测,将检测到的猕猴阳性标本扩增片段进行克隆测序。结果利用保守区域Rh85设计的引物可对人HCMV阳性对照进行扩增。用此方法检测的20份猕猴全血标本,出现2例阳性。其中一例扩增片段经纯化、回收克隆测序后用BLAST软件进行同源性对比,与GenBank中报道的RhCMV序列基本相同。结论建立了从猴全血中直接检测猴RhCMV病毒DNA的敏感、特异的nested PCR方法。     

西部马脑炎病毒基因组序列的半套式RT-PCR检测

为进一步提高RT－PCR检测西部马脑炎病毒（WEE）病毒基因组方法的敏感性,采用半套式PCR扩增病毒基因组特异序列,首先采用逆转录法将病毒基因组RNA逆转录为cDNA,然后以此cDNA为模板,进行扩增。对扩增后电泳检查无可见DNA条带的产物进行半套式PCR;与此同时对扩增的循环数、Mg^ 浓度和退火温度等条件进行了优化,以进一步提高扩增的特异性。结果第一轮PCR未扩出特异笥片段的WEE病毒稀释度,其半套式扩增出特定大小的DNA产物;同时优化的条件提高了扩增产物的特异性。扩增产物约为190bp的单一DNA片段,其大小与预期的相一致,结果表明采用半套式RT－PCR方法检测WEE病毒的基因组序列的敏感性可提高100倍以上。     

利用生物条形码技术对蓝舌病毒VP7蛋白进行微量检测

目的：建立高灵敏检测蓝舌病毒VP7蛋白的生物条形码检测方法。方法：制备VP7蛋白的多抗及特异DNA链标记的金纳米颗粒探针（NP）和VP7蛋白单抗标记的磁性微球探针（MMP）,形成MMP-VP7蛋白-NP三明治复合物后,再利用去杂交将NP探针上标记的DNA链释放出来,通过PCR或芯片检测方法鉴定释放的DNA链,确定VP7蛋白的存在。结果：建立了蓝舌病毒VP7蛋白的生物条形码检测体系,检测灵敏度可达10fg/mL,为常规ELISA检测的106倍。结论：为发展高灵敏度的蓝舌病毒生物条形码检测试剂盒鉴定了基础。     

四种广普性植物病毒高效mPCR检测方法的建立

本研究建立了能同时检测出烟草花叶病毒(TMV)、黄瓜花叶病毒(CMV)、马铃薯X病毒(PVX)和马铃薯Y病毒(PVY)的多重RT-PCR体系。TMV、CMV、PVX、PVY是四种广普性植物病毒,寄主范围广泛,并且常常发生复合侵染。本研究以上述四种病毒的CP基因部分序列设计引物,以反转录的cDNA为模板,建立多重RT-PCR反应体系,分别扩增出211～417bp的不同长度的基因片断,并通过序列测定来确认扩增序列的特异性。将反转录合成的cDNA进行浓度稀释,来对多重RT-PCR与单重RT-PCR的灵敏度进行比较,结果证明,多重RT-PCR体系能够同时快速检测这四种病毒,并且有很高的灵敏度。     

Plant infection by two different viruses induce contrasting changes of vectors fitness and behavior

Insect-vectored plant viruses can induce changes in plant phenotypes,thus influencing plant-vector interactions in a way that may promote their dispersal according to their mode of transmission (i.e.,circulative vs.noncirculative).This indirect vector manipulation requires host-virus-vector coevolution and would thus be effective solely in very specific plant-virus-vector species associations.Some studies suggest this manipulation may depend on multiple factors relative to various intrinsic characteristics of vectors such as transmission efficiency.In anintegrative study,we tested the effects of infection of the Brassicaceae Camelina sativa with the noncirculative Cauliflower mosaic virus (CaMV)or the circulative Turnip yellows virus (TuYV)on the host-plant colonization of two aphid species differing in their virus transmission efficiency:the polyphagous Myzus persicae,efficient vector of both viruses,and the Brassicaceae specialist Brevicoryne brassicae,poor vector of TuYV and efficient vector of CaMV.Results confirmed the important role of virus mode of transmission as plant-mediated effects of CaMV on the two aphid species induced negative alterations of feeding behavior (i.e.,decreased phloem sap ingestion)and performance that were both conducive for virus fitness by promoting dispersion after a rapid acquisition.In addition,virus transmission efficiency may also play a role in vector manipulation by viruses as only the responses of the efficient vector to plant-mediated effects of TuYV,that is,enhanced feeding behavior and performances,were favorable to their acquisition and further dispersal.Altogether,this work demonstrated that vector transmission efficiency also has to be considered when studying the mechanisms underlying vector manipulation by viruses.Our results also re- inforce the idea that vector manipulation requires coevolution between plant,virus and vector.     

植物组织粗汁液中的番木瓜环斑病毒的ELISA检测技术

本研究建立和改进了检测番木瓜和西葫芦组织粗汁液里的番木瓜环斑病毒（ＰＲＶ）的ＤＡＣ－ＥＬＩＳＡ法和Ｄｏｔ－ＥＬＩＳＡ法。用不同的ＥＬＩＳＡ方法来检测不同寄主植物粗汁液里的ＰＲＶ,其所用的合适的制备粗汁液的缓冲液是不同的。用ＤＡＣ－ＥＬＩＳＡ法检测西葫芦粗汁液时,以０．５ｍｏｌ／Ｌ磷酸盐缓冲液（ｐＨ７．５,内含０．１ｍｏｌ／Ｌ乙二胺四乙酸二钠）为宜;而检测番木瓜粗汁液时,则还要加入０．２５ｍｏｌ／Ｌ脲。用Ｄｏｔ－ＥＬＩＳＡ法检测时,在上述磷酸盐缓冲液中加入２％聚乙烯吡咯烷铜能提高对西葫芦粗汁液的检测效果。应用合适的制备粗汁液的缓冲液,ＤＡＣ－ＥＬＩＳＡ法和Ｄｏｔ－ＥＬＩＳＡ法的灵敏度分别提高到１／４０９６和１／１０２４（稀释度）。本研究还表明,影响ＤＡＣ－ＥＬＩＳＡ法的定过测定的主要因素是粗汁液的稀释度和包被液（０．０５ｍｏｌ／Ｌ碳酸盐缓冲液,ｐＨ９．６）的用过。在较高粗汁液稀释度和包被液的用量相同时,粗汁液里的病毒含量与ＤＡＣ－ＥＬＩＳＡ法的ＯＤ４９２ｎｍ值呈真实的线性关系。     

应用热平衡法测定玉米/大豆间作群体内作物的蒸腾量

通过田间试验采用基于热平衡法的茎流计测定玉米/大豆条带间作群体内作物的蒸腾规律.结果表明：间作群体内,玉米和大豆植株的茎流速率在晴天呈单峰曲线,在阴天则呈多峰曲线.植株的茎流受多个环境因子的影响,其中太阳辐射是影响植株茎流最主要的气象因子.玉米和大豆的单株日茎流量与多个气象因子间存在较好的相关关系,达到极显著水平.茎流观测期内（2008年6月1-30日）,间作群体内玉米植株的日均蒸腾量（1.44 mm·d^-1）为大豆（0.79 mm·d^-1）的1.8倍,玉米和大豆植株的蒸腾量分别占间作群体总蒸腾量的64%和36%.考虑到作物的茎直径和叶面积的空间变异,安装一定数量的茎流探头对于准确测定植株茎流是十分必要的.     

Inorganic Pyrophosphatase from E. coli as a Label for the Detection of Plant Viruses by ELISA

Inorganic pyrophosphatase (PPase) from E. coli was used as label in enzyme immunoassay for the detection of different plant viruses. The use of PPase-conjugated antibodies and tetrasodium pyrophosphate as a substrate in ELISA provides some advantages in detection of plant viruses in purified preparations and extracts from plant specimens: high sensitivity, negligible level of background reactions in control samples, bright blue-greenish colour which allows to detect the reaction visually and high stability of PPase-conjugated antibodies.     

The Occurrence of Translocated Antibiotics in Expressed, Plant Sap

The details of a convenient laboratory press are presented anda procedure by which plant sap is readily obtained is described.The pressing procedure has been shown to release 53–74per cent. of the total plant water as expressed sap. Preliminary observations on the uptake and translocation ofantibiotics by higher plants obtained by examining expressedsap are described. It is shown that the assay of expressed sapprovides a measure of the griseofulvin and chloramphenicol concentrationin leaves of plants grown in solutions of these antibioticsthat does not differ significantly from the value obtained bythe assay of water extracts. Extraction of leaves from plants grown in griseofulvin solutionswith organic solvents demonstrates a much greater antibioticcontent than is indicated by the assay of expressed sap. Toexplain this difference a ‘free’ antibiotic fraction,obtainable in expressed sap or water extracts, and a ‘bound’fraction recovered from the leaf only by organic solvent extractionare postulated.     

Improvement of antigen blotting in a tissue blot immunobinding assay for the detection of two chili pepper viruses

The tissue blot immunobinding assay (TBIA) is widely used for the detection and localization of plant viruses in various plant tissues. The basic experimental procedures of TBIA sampling and blotting were simplified using commercially available micropipette tips. This method was termed the ring-blot immunobinding assay (R-BIA), as the blot on the membrane forms a ring shape. The detection efficacy of R-BIA was tested for two chili pepper viruses, pepper mild mottle tobamovirus (PMMoV) and pepper mottle potyvirus (PepMoV), following the optimized serological procedures of TBIA (length of the incubation period and BSA concentration, and primary and secondary antibodies). Sensitivity of the R-BIA was about 1 ng/ml of purified PMMoV in pepper leaf sap from a healthy pepper plant. R-BIA also showed high specificity in the detection of PMMoV and PepMoV. Moreover, the modified sampling and blotting procedures were simpler and more reliable than other TBIA methods (such as whole-leaf blotting and crushed-leaf blotting), suggesting that the R-BIA may be used for medium- to large-scale detection of plant viruses in laboratories with minimal facilities.