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。     

小麦矮缩病毒外壳蛋白基因的原核表达、抗体制备及应用

 小麦矮缩病毒(Wheat dwarf virus,WDV)引起的小麦矮缩病是近年来我国小麦生产中的一种重要病毒病害,急需研发快速精准的检测技术用于预测预报和病毒-介体相互作用的研究。本研究应用Gateway重组技术构建了外壳蛋白基因(Coat protein, CP)的原核表达载体,将重组表达载体转化大肠杆菌Rosetta,经IPTG诱导获得CP基因原核表达蛋白。以重组蛋白为抗原免疫新西兰大白兔制备得到了相应的抗体,Western blot检测表明制备的抗体能与CP重组蛋白、感病小麦和带毒叶蝉特异性结合,说明获得的抗体特异性高。用获得的抗体进行免疫荧光标记,观察到病毒分布在介体叶蝉的前中肠和中中肠部位,为WDV的预测预报和介体条沙叶蝉传毒机制的研究奠定了基础。     

LNA探针实时荧光定量PCR检测小麦矮缩病毒体系的建立

 本研究通过对多个小麦矮缩病毒(Wheat dwarf virus, WDV)小麦分离物进行序列分析,在编码Rep蛋白N 端的基因保守区域设计一对特异性引物和一条长度为19 bp的TaqMan LNA探针,经过探针和引物浓度的系列优化,建立了WDV田间样品LNA探针实时荧光定量PCR检测方法,确定最优反应条件下的引物和探针终浓度分别为0.5 μmol/L和0.3 μmol/L。该方法建立的标准曲线斜率及相关系数分别为-3.424 3和0.997 3,扩增效率为96.0%,重复性试验组内变异系数为0.11%~1.34%,组间变异系数为0.44%~1.21%,最低检测限为55 copies/μL,其灵敏度是普通PCR的100倍以上。与普通TaqMan探针荧光定量PCR相比,该方法所用探针长度大大缩短,减少了探针间形成二级结构的机会,探针设计更加简单、方便。该方法适用于田间样品的早期监测,为病害流行调查提供了高效快捷的技术支撑。     

马铃薯类病毒(PSTVd)非放射性标记cDNA探针制备及其在检测上的应用

 利用含PSTVd单体克隆的重组质粒pGEM PSTVd,通过PCR扩增技术,用生物素标记制备cDNA探针,进行杂交反应检测PSTVd,其中通过化学颜色反应进行判读灵敏度可达50pg,而用化学发光反应进行判读灵敏度可达5pg,分别是R-PAGE检测灵敏度的26倍和260倍,且2种反应特异性和专化性较强。cDNA核酸斑点杂交反应(NASH)检测PSTVd方法准确、灵敏度高,一次检测样品数量多,且对异地样品检测非常方便,是以往其它检测方法的有效补充。     

福建马铃薯S病毒的分子鉴定及发生情况

为明确福建省马铃薯S病毒(PVS)的发生与分布情况,对福建省马铃薯主要种植区的PVS进行了鉴定和普查.在利用电镜技术和传统生物学方法鉴定的基础上,克隆了PVS外壳蛋白(cp)基因,依据PVS外壳蛋白氨基酸序列建立了PVS不同分离物的系统进化树.研究表明,利用PVS 外壳蛋白氨基酸序列分析可准确鉴定PVS,同时可分析不同分离物间的分子差异.利用病毒特异性引物和DIG标记的PVS cp基因为探针,分别利用RT-PCR技术和核酸斑点杂交技术(NASH)对PVS进行了检测,并对检测技术进行了改进.调查结果表明,PVS在福建省广泛分布,发病率最高可达80%以上,当地农家自留种可能是田间PVS的主要来源.     

辣椒轻斑驳病毒葫芦岛分离物RNA杂交检测体系的建立

辣椒轻斑驳病毒(pepper mild mottle virus,PMMoV)可引起辣椒叶片以及果实的花叶和畸形症状,造成相当的经济损失。为了建立辣椒轻斑驳病毒的高特异性和灵敏度的分子检测体系,采用非放射性的化合物地高辛(DIG)标记检测PMMoV正义链的RNA探针,建立了该病毒Dot blot杂交和Northern blot杂交检测体系,并通过RT-PCR法验证了杂交体系的检测特异性。结果表明,RNA探针对PMMoV具有很高的检测特异性和灵敏度,适用于病毒的早期检测以及相关分子研究。     

应用常规RT-PCR和荧光定量RT-PCR检测柑桔衰退病毒

 根据柑桔衰退病毒(CTV)P20基因序列设计cquctv9/cquctv10特异引物对,以柑桔RNApolymeraseⅡ基因作为内参照,建立了柑桔衰退病的常规RT-PCR快速检测体系;依据柑桔衰退病毒P20基因序列设计cquctv1/cquctv2特异引物对和TaqMan探针cquctvp1,建立了柑桔衰退病荧光定量RT-PCR快速检测体系。常规RT-PCR检测下限是含有CTV的50pg总RNA,荧光定量RT-PCR法的检测下限是2fg纯CTV片段。荧光定量RT-PCR的灵敏度相对比常规RT-PCR高100倍。利用常规RT-PCR和荧光定量RT-PCR体系对从2005年3月到2006年7月采自田间的样品进行检测,结果表明,2种检测体系都有很好的特异性和准确性;对183个田间柑桔苗木样品带毒率检测结果表明,荧光定量RT-PCR检出率为(82.5%),比常规RT-PCR检出率(73.2%)高。     

五种烟草病毒TMV、CMV、TEV、PVY及TVBMV的多重RT-PCR同步检测

 我国烟草病毒主要有烟草花叶病毒(TMV)、黄瓜花叶病毒(CMV)、烟草蚀纹病毒(TEV)、马铃薯Y病毒(PVY)和烟草脉带花叶病毒(TVBMV),通常发生复合侵染。本研究对我国5种烟草病毒的外壳蛋白基因部分序列设计引物,通过优化引物和模板浓度,摸索扩增参数,在一个体系中成功对5种病毒复合侵染的烟草材料进行多重RT-PCR扩增,得到237、273、347、456和547 bp共5条特异性条带,建立了能同时检测TMV、CMV、TEV、PVY和TVBMV的多重RT-PCR检测体系。对田间样品检测结果证明,多重RT-PCR体系能够同时检测5种病毒,并且灵敏度高。     

实时荧光PCR检测瓜炭疽病菌

采用实时荧光PCR技术建立了瓜炭疽病菌(Colletotrichum orbiculare)的检测方法。根据瓜炭疽病菌甘油醛-3-磷酸脱氢酶(GAPDH)基因和谷氨酰胺合成酶(GS)基因序列,设计了该病菌特异性引物和TaqMan探针,并对所设计的引物和探针的反应条件进行了优化。采用本试验建立的实时荧光PCR方法对瓜上的其他菌株及近似菌株进行检测,可将瓜炭疽病菌与其他病原菌区分开。灵敏度试验表明,25μL体系中只要有39.6pg的核酸量就可以被检测到,检测灵敏度达到1.584pg/μL,比普通PCR检测灵敏度高100倍。同时对田间采集的病株和未知样品进行的检测证明了引物和TaqMan探针的特异性。     

抗草甘膦转基因大豆Cp4-epsps基因快速简便的LAMP检测方法

针对抗草甘膦转基因大豆的外源基因Cp4-epsps,建立了一种基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术的抗草甘膦转基因大豆的检测体系,其扩增产物既可利用常规琼脂糖凝胶电泳检测,还可通过SYBR Green I染色进行快速检测。LAMP检测体系中dNTPs浓度为0.8 mmol/L、Mg2+浓度为3mmol/L、反应时间为45min时扩增效果最佳,其检测灵敏度为5μg/L,比常规PCR灵敏100倍。田间实际检测结果表明,LAMP检测结果和PCR检测结果完全一致,准确率为100%。本研究所建立的抗草甘膦转基因大豆LAMP检测方法具有简便快速、特异性强、灵敏度高等特征,是一种能够用于抗草甘膦转基因大豆检测、田间基因漂移监测和环境安全研究的有力工具。     

麦长管蚜OBP7基因的RNA干扰

OBP是蚜虫嗅觉功能中的一类重要分泌蛋白,能选择性地结合气味分子并进行信号转导。本试验使用显微注射法将麦长管蚜OBP7的小干扰RNA(siRNA)导入麦长管蚜体内,通过qRT-PCR检测OBP7mRNA相对表达量的变化情况。结果发现在siRNA浓度0.32μg/μL,注射量23nL,注射后24h,麦长管蚜成蚜的mRNA相对表达量降低到37.1%,证明了显微注射方式进行RNA干扰的可行性。在注射23nL的siRNA-467,经过36h后,麦长管蚜出现了对EβF趋性行为的改变(P0.05),表明OBP7蛋白在麦蚜对EβF的识别过程中具有重要作用。     

陕西韩城严重发生的小麦矮缩病病原鉴定与原因分析

2007年在陕西韩城发现一种新的小麦病毒病害,症状表现为严重矮缩、黄化、条斑和分蘖增多等,发病面积约0.07万hm²,病田减产达50%,严重地块甚至绝收。本研究通过对采集自我国陕西韩城的7个样品进行PCR鉴定、全基因组序列测定及比较,证实陕西韩城样品确是小麦矮缩病毒(WDV)侵染所致,并对发病原因及其流行趋势进行了分析。这是小麦矮缩病在我国麦田大发生的首次报道。     

Partial resistance to Wheat dwarf virus in winter wheat cultivars

Four Hungarian winter wheat cultivars were investigated for their susceptibility to the geminivirus Wheat dwarf virus (WDV). Previously, two cultivars (Mv Regiment and Mv Emese) were assessed by breeders to exhibit virus symptoms in the field, whereas Mv Dalma and Mv Vekni showed few symptoms. Two inoculation techniques for WDV, vector transmission with the leafhopper Psammotettix alienus and agroinoculation, were used. Leafhopper transmission was more efficient than agroinoculation. However, irrespective of the technique used, no Mv Dalma or Mv Vekni plants showed clear WDV symptoms. In contrast, 3/30 Mv Emese and 4/36 Mv Regiment plants showed dwarfing and chlorosis after agroinoculation and 13/17 and 14/15 plants, respectively, had clear WDV symptoms after vector transmission. WDV‐specific PCR showed that Mv Vekni and Mv Dalma plants could be infected, especially following vector transmission (approximately 50% infection), but at significantly lower frequency than Mv Emese or Mv Regiment plants (100% infection). Furthermore, real‐time PCR showed that WDV DNA accumulated to much lower levels in infected Mv Vekni and Mv Dalma plants than in infected Mv Regiment and Mv Emese plants. The data strongly suggest that Mv Vekni and Mv Dalma are partially resistant to WDV infection. As WDV resistance has not previously been identified in wheat, and because WDV can cause significant yield losses, the resistance of Mv Vekni or Mv Dalma will provide a valuable breeding resource.     

Sequence analyses of Wheat dwarf virus isolates from different hosts reveal low genetic diversity within the wheat strain

Wheat dwarf virus (WDV) causes disease in wheat (Triticum aestivum) and barley (Hordeum vulgare) in many parts of Europe. The host range also includes many species of the family Poaceae. WDV is only transmitted by the leafhopper Psammotettix alienus. During a five‐year period (2001–2005), grass samples were collected in central Sweden in the vicinity of fields with WDV‐infected winter wheat. Screening with ELISA and PCR identified WDV in a low number of samples (8/1098) from only three grass species: Apera spica‐venti, Avena fatua and Poa pratensis. In addition, triticale was found to be positive. Fourteen WDV isolates from Avena fatua, Apera spica‐venti, Triticum aestivum, Lolium multiflorum, Poa pratensis, triticale and the insect vector Psammotettix alienus, were partially sequenced (ca. 1200 nucleotides), providing the first published WDV sequences from the insect vector. All isolates belonged to the wheat strain of WDV and the genetic diversity was low. Phylogenetic analyses showed no clear grouping according to geographical location or host species. The results suggest that the same WDV genotypes are infecting both wheat and grasses in Sweden. Interestingly, one group of isolates (subtype B) formed a distinct clade in the phylogenetic tree. Subtype B was always found in mixed infection with the main genotype. Complete sequencing of a subtype B isolate showed that it was 98·6% identical to a typical wheat isolate from the same plant.     

Reservoirs of plant virus disease: Occurrence of wheat dwarf virus and barley/cereal yellow dwarf viruses in Sweden

Non-crop plants such as grasses and volunteer plants are an inseparable part of the flora of crop fields and can influence virus incidence in crop plants. The presence of grasses as virus reservoirs can lead to a higher probability of virus incidence in crop plants. However, the role of reservoirs as an inoculum source in agricultural fields has not been well studied for many viral diseases of crops. Grasses have been found to constitute potential reservoirs for cereal-infecting viruses in different parts of the world. This study revealed that cereal-infecting viruses such as wheat dwarf virus (WDV), barley yellow dwarf viruses (BYDVs), and cereal yellow dwarf virus-RPV (CYDV-RPV) can be found among ryegrass growing in or around winter wheat fields. Phylogenetic analysis showed that a WDV isolate from ryegrass was a typical WDV-E isolate that infects wheat. Similarly, a ryegrass isolate of barley yellow dwarf virus-PAV (BYDV-PAV) grouped in a clade together with other BYDV-PAV isolates. Inoculation experiments under greenhouse conditions confirmed that annual ryegrass of various genotypes can be infected with WDV to a very low titre. Moreover, leafhoppers were able to acquire WDV from infected ryegrass plants, despite the low titre, and transmit the virus to wheat, resulting in symptoms. Information from the grass reservoir may contribute to improving strategies for controlling plant virus outbreaks in the field. Knowledge of the likely levels of virus in potential reservoir plants can be used to inform decisions on insect vector control strategies and may help to prevent virus disease outbreaks in the future.     

Occurrence of viruses in irrigated wheat in Zambia

Surveys were conducted during the cool-dry months of June–August 1997 and June–July 1998 for the presence of viruses in irrigated wheat in Central, Copperbelt, Lusaka and Southern Provinces of Zambia in 14 commercial farms and four wheat cultivar plots. Virus symptoms were observed on nine wheat cultivars ( Triticum aestivum 'Deka', 'Gamtoos', 'Lorie II', 'MM2', 'Nata', 'Nkwazi', 'P7', 'Scan' and 'Sceptre') of South African, Zambian and Zimbabwean origin. Several viruses were identified on the basis of field symptomatology, symptoms developing on mechanically inoculated indicator plant species or cultivars and serology (DAS-ELISA). The study revealed the occurrence of Brome mosaic virus (BMV), Barley stripe mosaic virus (BSMV), Barley yellow dwarf virus and its strains (BYDV-PAV and RPV), Soil-borne wheat mosaic virus (SBWMV), Wheat dwarf virus (WDV), Wheat streak mosaic virus (WSMV) and Wheat spindle streak mosaic virus (WSSMV). DSA-ELISA tests confirmed these identifications. The prevalence of viruses varied annually and from field to field. BSMV, BYDV-PAV, SBWMV, WDV, WSMV and WSSMV were found to be the most prevalent viruses. Viruses generally occurred in mixed infections of 3–6 viruses and the most common virus complex consisted of 4 viruses (50%), viz. BYDV, SBWMV, WDV and WSSMV. Five- and six-virus complexes were relatively less common (20% each) whereas 3-virus complex was noticed in only 10% cases. SBWMV and WSSMV have been found to be new to Africa and Zambia and are reportedly vectored by a fungal protist – Polymyxa graminis . BYDV strains MAV and SGV were also tested but gave negative results against their antisera.     

Studies on the host range of the barley strain of Wheat dwarf virus using an agroinfectious viral clone

Comparative analysis of the host ranges of the barley and wheat strains of Wheat dwarf virus (WDV; family Geminiviridae ; genus Mastrevirus ) in Europe has been severely hampered by the lack of an infectious clone of the barley strain. To remedy this situation an agroinfectious clone of a Hungarian isolate of the barley strain (WDV-Bar[HU]) was constructed and its virulence tested in barley ( Hordeum vulgare ), wheat ( Triticum aestivum ), rye ( Secale cereale ) and oat ( Avena sativa ) by agroinoculation. Although all four species could be systemically infected by the isolate, infections were asymptomatic in the rye and oat cultivars tested. WDV-Bar[HU] induced chlorosis and stunting symptoms typical of WDV in barley, while in wheat low infection rates but high mortality of infected seedlings were observed. In contrast, a much higher percentage of wheat plants agroinoculated with a wheat-strain isolate (WDV-[Enk1]) became systemically infected. WDV-[Enk1] in wheat caused symptoms similar to those caused by WDV-Bar[HU] in barley. WDV-Bar[HU] was leafhopper-transmissible to barley seedlings, in which it caused typical WDV symptoms; geminate virus particles were isolated from the infected leaves. Comparison of the genomic sequences of 11 barley strain isolates from Europe and Turkey revealed that whereas WDV-Bar[HU] represents a typical barley-strain isolate that is not detectably recombinant, the Turkish barley isolate (WDV-Bar[TR]) is probably a recombinant between a barley-strain isolate and an as-yet-undescribed WDV-like mastrevirus species.