稻曲病菌薄壁分生孢子制备的影响因素分析

本文研究了培养基、温度、振荡速度等因素对稻曲病菌Ustilaginoidea virens薄壁分生孢子产孢量的影响。结果表明,稻曲病菌薄壁分生孢子在培养第7天基本达到最大孢子量;该菌最适宜产孢的培养基为马铃薯煮汁,在煮汁中添加蔗糖可大幅提高产孢量;适宜的产孢温度为26~28℃;静止培养不利于产孢,振荡培养有利于产孢,并表现为转速越高产孢量越多;光照条件对产孢量没有影响。     

3种甘薯病毒多重RT-PCR检测方法的建立及应用

正病毒病是引起甘薯品质降低和减产的重要原因之一,现已报道30多种能侵染甘薯的病毒~([1,2])。山东省是甘薯种植大省,病毒种类近10种~([3,4])。甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFM V)、甘薯潜隐病毒(Sweet potato latent virus,SPLV)是为害甘薯的主要病毒,在全国甘薯种植区广泛分布~([5,6])。甘薯病毒2(Sweet potato virus 2,SPV2)为Potyvirus的一个暂定种,多与同属的其他病毒混合侵染~([7])。多重PCR技术由Chamberian等~([8])1988年首次提出,可实现多基因的同时扩增,具有节省时间、提高效率的优点,已初     

利用多重RT-PCR技术检测草莓病毒的研究

 目前已报道侵染草莓的病毒有20多种,其中草莓斑驳病毒(Strawberry mottle virus, SMoV)、草莓轻型黄边病毒(Strawberry mild yellow edge virus, SMYEV)和草莓镶脉病毒(Strawberry vein banding virus, SVBV)是分布较广、危害较重的3种主要病毒。     

Detection of four calla potyviruses by multiplex RT-PCR using nad5 mRNA as an internal control

Dasheen mosaic virus (DsMV), Turnip mosaic virus (TuMV), Konjac mosaic virus (KoMV) and Zantedeschia mild mosaic virus (ZaMMV) are important potyviruses previously identified in calla lily plants in Taiwan. In order to save time and cost of virus detection, a multiplex RT-PCR assay was developed for these calla potyviruses. Specific primers for each virus were designed based on the sequences of 3′ terminal region of respective viruses. To prevent false negative results, a primer pair specific to plant mitochondrial nad5 mRNA was used to produce a 185-bp fragment as an internal control of RT-PCR. The specificities of primers were confirmed by means of simplex and multiplex PCR assays. Optimal primer concentration ratio was identified by multiplex PCR assay. Total RNAs purified from virus-infected plants were used directly or mixed in different combinations, and then tested by multiplex RT-PCR. The result indicated that the expected RT-PCR products could be specifically amplified and identified on the basis of their molecular sizes. The detection sensitivity of multiplex RT-PCR was 25–625 times higher than that of indirect-ELISA (I-ELISA) depending on the virus. When applied to field surveys, multiplex RT-PCR could detect more single as well as mixed infection samples than I-ELISA. Accordingly, our multiplex RT-PCR assay provides a simple, rapid and reliable method for multiple potyvirus detection in calla lily.     

A more sensitive and rapid multiplex RT-PCR assay combining with magnetic nanobeads for simultaneous detection of viruses in sweet potato

An improved multiplex RT-PCR assay combined with magnetic nanobeads (MNB-RT-PCR) was developed for simultaneous detection of four sweet potato viruses, Sweet potato virus G (SPVG), Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC) and Sweet potato chlorotic fleck virus (SPCFV). Four primer pairs specific for each virus were designed and the corresponding PCR products were 169, 357, 516 and 900 bp in length for SPVG, SPFMV, SPVC and SPCFV, respectively. The specificity of the method was tested using different combinations of virus templates, and the identities of the amplification products were confirmed by sequencing. The limits of detection for all four viruses by single and multiplex MNB-RT-PCR assays were comparable. The assay was further evaluated using laboratory and field samples compared with a conventional CTAB-RT-PCR assay, and the comparative results showed that the MNB-RT-PCR assay was more rapid and sensitive. These results suggest that the multiplex MNB-RT-PCR assay is an effective and preferable method for virus detection in sweet potato.     

基于小RNA深度测序和RT-PCR检测侵染广东省冬种马铃薯的病毒

为明确侵染广东省冬种马铃薯的病毒种类及优势病毒,结合小RNA深度测序技术及RTPCR检测方法,对采集于广东省冬种马铃薯7个主产区的189份疑似病样进行检测分析。结果表明,经小RNA深度测序技术检测马铃薯病毒病混合样,发现存在马铃薯Y病毒(Potato virus Y,PVY)、马铃薯S病毒(Potato virus S,PVS)和马铃薯卷叶病毒(Potato leaf-roll virus,PLRV)3种病毒。进一步设计3种病毒的特异性引物并利用国内已报道的其它5种马铃薯病毒的特异性引物进行RT-PCR检测,发现189份马铃薯病毒病疑似病样中仅检测到PVY、PVS和PLRV这3种病毒,检出率依次为75.13%、10.05%和4.76%,且3种病毒在马铃薯上还存在复合侵染,复合侵染率为14.19%,其中PVY在各马铃薯产区均可检测到。表明侵染广东省冬种马铃薯的病毒为PVY、PVS和PLRV,其中PVY是优势病毒。     

Detection of carrot red leaf virus-RNA in carrot seeds by multiplex RT-nested PCR

A practical method to detect carrot red leaf virus (CtRLV)-RNA in carrot seeds using a multiplex RT-nested PCR was developed. Total RNA was extracted from carrot seeds by the guanidine thiocyanate method. RT-PCR was performed with a one step RT-PCR kit with CtRLV primers and carrot ubiquitin primers for the internal control, and the second PCR was performed with CtRLV nested primers. Ubiquitin were detected by the first PCR in all samples, while distinct CtRLV-RNA was detected by the 2nd PCR. CtRLV-RNA was detected from carrot seeds from three cultivars. The multiplex RT-nested PCR method was able to detect CtRLV-RNA from a single carrot seed.     

侵染西瓜的5种病毒ZYMV、WMV、TMV、SqMV和CMV的多重RT-PCR检测体系的建立与检测应用

 根据5种病毒小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)、西瓜花叶病毒(Watermelon mosaic virus,WMV)、烟草花叶病毒(Tobacco mosaic virus,TMV)、南瓜花叶病毒(Squash mosaic virus,SqMV)和黄瓜花叶病毒(Cucumber mosaic virus,CMV)的核苷酸保守区序列,设计特异性引物对,从影响多重RT-PCR (mRT-PCR)扩增的引物浓度、Mg²⁺浓度、Taq DNA聚合酶浓度、dNTPs浓度、退火温度等方面进行反应体系的优化,建立了一种能够同时检测ZYMV、WMV、TMV、SqMV和CMV的多重RT-PCR技术体系,并进行了实际应用。在一个体系中对上述5种病毒复合侵染的西瓜材料进行多重RT-PCR扩增,得到与试验设计相符的5条特异性条带,依次是542、485、410、354和293bp。该体系实现了对侵染西瓜的5种病毒的同时检测,极大地提高了检测效率,降低了检测成本,体现了多重RT-PCR的优越性。     

4种番茄病毒多重RT-PCR检测及应用

<正>番茄是全世界栽培最为普遍的果菜之一,2011年世界番茄栽培面积约805万亩,年产量约3 773万t,我国是世界番茄种植大国之一,2011年面积96667公顷,产量约679万t,随着番茄种植面积不断扩大,番茄病毒病的危害逐年加重~([1])。世界范围内番茄除了已有的TMV抗病资源与育成的抗     

马铃薯4种病毒多重PCR检测体系的建立

我国马铃薯病毒主要有马铃薯Y病毒(PVY)、马铃薯X病毒(PVX)、马铃薯S病毒(PVS)、马铃薯卷叶病毒(PLRV),常发生复合侵染。根据GenBank中4种马铃薯病毒的外壳蛋白(coat protein,CP)基因全长设计引物,通过RT-PCR扩增得到4种病毒CP基因全长片段,测序结果显示序列同源性96%以上;针对4种病毒CP基因的保守序列分别设计引物,在一个PCR体系中同步对4种病毒进行扩增,得到421、202、516、330bp的特异性条带,优化建立了能同步检测PVY、PVX、PVS和PLRV的多重RT-PCR检测体系。检测结果证明优化后的多重RT-PCR体系能在田间样品中快速、高效地检测出4种病毒。     

Simultaneous detection and identification of eight stone fruit viruses by one-step RT-PCR

A sensitive and reliable one step RT-PCR reaction with an internal control has been developed to detect and differentiate eight important viruses that affect stone fruit tress: Apple mosaic virus (ApMV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), American plum line pattern virus (APLPV), Plum pox virus (PPV), Apple chlorotic leaf spot virus (ACLSV), Apricot latent virus (ApLV) and Plum bark necrosis stem pitting associated virus (PBNSPaV). In addition, we investigated the detection limit and the efficiency of three different nucleic acid extraction methods that avoid the use of organic solvents, for both multiplex RT-PCR and dot-blot hybridisation assays. The primer cocktail was used to analyse 38 stone fruits originating from nine different countries and six species. A large number of virus combinations was detected and up to three different viruses were observed in five samples. A decrease in sensitivity was observed when the primer cocktail contained more than five different pair primers. However, comparative analyses showed that the multiplex RT-PCR containing the eight virus pair primers was even more sensitive than the ELISA or molecular hybridisation assays. The use of the multiplex RT-PCR technology in routine diagnosis of stone fruit tree viruses is discussed.