【作者】 杜志游；

【导师】 陈集双；

【作者基本信息】 浙江大学 ， 微生物学， 2006， 博士

【摘要】 自然侵染马铃薯作物的病毒超过25种。其中，部分病毒普遍发生且对马铃薯作物造成严重的危害，显著降低了马铃薯的产量和品质。据文献报道，在中国马铃薯产区，对马铃薯作物产生重要危害的病毒有马铃薯A病毒（PVA）、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）和马铃薯纺锤块茎类病毒（PSTVd）。本论文基于检测病毒的数目和检测样品的多少，建立四套适合不同检测需求的马铃薯病毒检测方法。一是：病毒粒子直接吸附-反转录-聚合酶链式反应（VDA-RT-PCR），适合少量样品的单种病毒检测；二是：多重RT-PCR，以18S核糖体RNA（rRNA）作为内参照，针对以上描述的5种重要病毒的同时检测；三是：寡聚核苷酸微点阵技术，针对7种马铃薯病毒和一种类病毒的同时检测；四是：玻片杂交方法，适合大量马铃薯样品的同时检测。本论文首先利用分子克隆方法对PVA、PVX、PVY、PVS、PLRV、烟草花叶病毒（TMV）、黄瓜花叶病毒（CMV）和PSTVd保守序列以及18S rRNA进行扩增、克隆和测序；并对测序结果进行同源性分析。同源性分析结果表明PVX与组Ⅱ株系的同源性高达94.0％-100.0％，而与组Ⅰ株系的同源性则只有83.0％-86.0％；可见，该分离物应属于组Ⅱ株系。CMV与亚组Ⅰ株系的同源性高达94.5％-98.6％，而与亚组Ⅱ株系的同源性则在75.7％-78.9％；可见，该分离物应属于亚组Ⅰ株系。然而，PVY病毒则未表现序列同源性与株系之间的联系，序列同源性呈连续分布（88.0％-100.0％），其它的病毒或PSTVd的同源性除个别序列外，均在94.0％以上。高同源性说明了以上几种病毒和类病毒的序列可以用于制备病毒、病毒株系和类病毒特异的探针。病毒粒子直接吸附—反转录—聚合酶链式反应（VDA-RT-PCR）该方法针对于少量样品的单个病毒检测。我们利用离心管壁对病毒粒子的非特异吸附特性，以PVX作为研究材料，建立一种简单、快速、无需酚氯仿抽提制备马铃薯病毒RNA的方法，该方法被称为病毒粒子直接吸附法（Virion-direct absorbent，VDA）。结合RT-PCR反应对VDA制备的病毒RNA进行检测。一系列条件试验结果表明：利用普通提取缓冲液、1hr孵育时间和70℃变性吸附于管壁的病毒粒子可以获得最优化的病毒RNA制备方法。吸附于管壁的病毒粒子在室温的储存期限高达11天。优化的VDA-RT-PCR方法同样适合于PVY和PVS检测。灵敏度测试结果表明我们建立的VDA-RT-PCR法检测PVX的最低极限为1.25 ng，相当于ELISA检测的水平。尽管该方法的检测灵敏度不高，但是，该方法非常简单、快速和经济，单管即可完成病毒RNA的提取。多重RT-PCR该方法利用18S rRNA作为内参照，针对5种重要马铃薯病毒的同时检测。一些报道利用了植物的NADH脱氢酶mRNA作为内参照来监控核酸提取和RT-PCR反应的成功与否。然而，在real-time RT-PCR体系中，18S rRNA也常被用作内参照。为此，为了获得更合适的内参照，我们比较了寄主的nad2 mRNA和18S rRNA二者的检测最低极限和降解动力学。检测后者的最低极限是前者的10⁵倍，这说明18S rRNA作为内参照具有更高的灵敏度；而且，与PVX相比，18S rRNA比nad2 mRNA表现更加相似的降解动力学属性。为此，我们选择了18S rRNA作为一套同时检测5种重要马铃薯病毒的多重RT-PCR反应体系的内参照。通过一系列的优化试验，结果表明在标准RT-PCR反应体系的基础上，改变Taq HS DNA聚合酶的浓度为0.1 U／μl和MgCl₂浓度为4.0 mM，可以获得最优化的、能同时扩增PLRV、PVY、PVX、PVA、PVS和18S rRNA目的片段的多重PCR反应体系。优化的多重RT-PCR反应体系能扩增不同的病毒组合。该体系检测PVX的灵敏度是DAS-ELISA方法的100倍，前者在后者未检测到病毒的几个样品中检测到PVX和PVS，这也说明了优化的多重RT-PCR反应体系比DAS-ELISA具有更高的检测灵敏度。寡聚核苷酸微点阵技术该方法针对7种马铃薯病毒和一种类病毒的同时检测。目前，已有文献报道DNA微点阵技术应用于马铃薯病毒的检测。这些微点阵技术利用反转录标记方法使得它们的检测灵敏度均偏低，这是这些DNA微点阵技术的缺陷所在。因此，我们试图利用多重PCR标记方法和25 mer左右的寡聚核苷酸探针，建立高灵敏、高特异、同时检测7种马铃薯病毒和PSTVd的微点阵技术。研究结果表明：我们构建的寡聚核苷酸微点阵体系特异性很强，标记产物与异源探针不发生任何的交叉杂交；通过对两组多重标记PCR的优化，能同时标记PLRV、PVY、PVX、PVA、PVS、CMV、TMV、PSTVd和18S rRNA。我们构建的寡聚核苷酸微点阵结合优化的多重标记PCR方法可以检测10^-9倍数稀释的CMV，而核酸斑点杂交技术检测的最低极限为10^-5或10^-6，可见，多重标记PCR方法的介入显著提高了寡聚核苷酸微点阵技术的检测灵敏度。我们建立的寡聚核苷酸微点阵技术的高灵敏度显著高于其它马铃薯病毒检测微点阵技术。玻片杂交该方法适合于同时检测大量马铃薯样品，具备高通量检测的特点。DNA微点阵技术作为诊断工具，它的优点在于能同时对多个病原物进行检测，但是，该技术并不适合于从大量样品中检测个别病毒。为了解决这个问题，我们试图将从马铃薯组织中提取的总RNA样品直接点在氨基硅烷玻片表面，用CY5标记的探针进行杂交，建立一套同时检测大量马铃薯样品的玻片杂交方法，来替代常规的RNA核酸斑点杂交（NASH）。条件试验结果表明适合于尼龙膜杂交的甲酰胺点样液和DMSO点样液并不适合于氨基修饰的玻片，其对RNA在氨基玻片表面的结合效率显著低于SSC缓冲液。高浓度SSC（5×）有利于RNA在玻片表面的结合，而相对低浓度SSC（1×）则表现显著的线性相关性。不同类型的玻片对RNA结合效率有着显著的差别，氨基硅烷玻片具有明显的优势，其产生的杂交信号显著高于多聚赖氨酸和醛基修饰的玻片。为此，我们选择了1×SSC作为点样缓冲液和氨基硅烷玻片作为RNA支持物，测定了玻片杂交的检测灵敏度。结果显示：PCR方法标记和随机引物方法标记的探针检测TMV RNA的极限分别为1.71 pg和13.67 Pg；而用³²p标记的探针和尼龙膜杂交检测TMV RNA的最低量为6.83 pg。尽管PSTVd特异探针与PVY侵染的心叶烟总RNA出现弱的交叉杂交，但是在PVA、PVY和ZYMV三者之间没有出现交叉反应，这说明该方法还是具有相当高的特异性。田间马铃薯样品的检测结果说明了该方法具有同时检测大量马铃薯样品的能力。每种诊断方法都有它的优点和不足之处，包括本论文建立的4套马铃薯病毒检测方法，比如说，用多重RT-PCR反应检测单种病毒，这样就显著增加了检测的成本，而用VDA-RT-PCR方法既可以节省成本（特别是RNA提取的试剂），又可以节省时间和劳动力。如果从大量马铃薯样品中检测少数几种病毒，我们应该选择玻片杂交方法，而不是寡聚核苷酸微点阵技术。如果从少数样品中检测多个病毒，我们应该选择多重RT-PCR或寡聚核苷酸微点阵技术，而不是玻片杂交。因此，我们可以根据不同的检测需求，有针对性地选择最为合适的诊断方法，使更多还原

【Abstract】 More than 25 viruses have been reported to naturally infect potato crops. Of them, some cause severe damage to the crop, which potentially decrease yield and quality of potato. As reported, viruses producing acute damage to the crop in native potato-producing areas have Potato virus A （PVA）, Potato virus Y （PVY）, Potato virus X （PVX）, Potato virus S （PVS）, Potato leaf roll virus （PLRV） and Potato spindle tuber viroid （PSTVd）. In this study, four sets of methods were developed for different requirements of detection of potato viruses according to numbers of potato viruses and test samples. The first method is virion-direct absorbent-reverse transcriptase-polymerase chain reation （VD A-RT-PCR）, designed for detection of few viruses in some samples; the second is multiplex RT-PCR combined with 18S rRNA as an internal control, designed for simultaneous detection of five important viruses above mentioned; the third is oligonucleotide microarray technique, designed for simultaneous detection of seven potato viruses and one viroid; the last one is glass slide nucleic acid hybridization, designed for detecting potato viruses or viroid from large numbers of potato samples in one reaction.First, cloning and sequencing for the conserved sequences of PVA, PVX, PVY, PVS, PLRV, tobacco mosaic virus （TMV）, cucumber mosaic virus （CMV）, PSTVd and 18S rRNA were perfomed according to molecular cloning method. Results of homology analyses showed that, the PVX isolate had a similarity of 94.0%-100.0% to group II strains, and 83.0%-86.0% to group I strains. The isolate should be classified to group II. The CMV isolate was 94.5%-98.6% identical to subgroup I strains, and 75.7%-78.9% identical to subgroup II strains. The isolate should be a strain of subgroup I. However, PVY did not have association between sequence homologies and strains. Other viruses or PSTVd had a similarity of up to 94.0% with few exceptions. These high homogies suggested that these sequences could be used to prepare virus-specific or strain-specfic probes for nucleic acid hybridization.Virion-direct absorbent-reverse transcriptase-polymerase chain reaction （VDA-RT-PCR）. This method was designed for detecting single virus from some potato samples. We attempted to utilize the character of non-specific absorption between test tube and viral particles to establish a rapid, simple, phenol-chloroform-free method for preparation of potato viral RNAs. This method was named ’virion-direct absorbent’. VDA combined with RT-PCR （VDA-RT-PCR） was used for detection of potato viruses. Results from a series of conditional assays showed that, optimal protocol was set up by utilizing general extraction buffer, incubation for one hour, and denaturing viral particles at 70°C. Absorbed virions could be preserved at room temperature for up to 11 days. The optimal VDA-RT-PCR protocol could detect 1.25 ng PVX, similarly sensitive to ELISA method. Although the protocol had not high sensitivity, it was very simple, timesaving and costsaving. Viral RNA could be prepared in only one tube.Multiplex RT-PCR. This method combined with 18S rRNA as an internal control, was designed for simultaneous detection of five important viruses. Some reports used a plant NADH dehydrogenase mRNA as an internal control for the success of RNA extraction and RT-PCR reaction. However, 18S ribosomal RNA （rRNA） usually be used as an internal control in real-time RT-PCR systems. Therefore, in order to search for a perfect internal control, the 18S rRNA was compared with the commonly used nad2 mRNA in terms of detection sensitivity and degradation kinetics. Detection limit of 18S rRNA was 5 magnitudes higher than that of nad2 mRNA, demonstrating that 18S rRNA used as an internal control was more sensitive. The 18S rRNA also displayed degradation kinetics more similar to that of PVX. Thus, 18S rRNA was used as an internal control in a multiplex RT-PCR system for simultaneous detection of five economically important potato viruses. By a series of optimization assays, optimal multiplex RT-PCR was achieved by increasing concentrations of Taq HS DNA polymerase and MgCl₂ to be 0.1 U/μl and 4.0 mM, respectively. The optimal multiplex RT-PCR system could simultaneously amplify cDNAs from PVA, PVX, PVY, PLRV, PVS, and 18S rRNA, and was able to detect all viruses under different combinations of the viruses. The technique was 100- fold greater for detection of PVX than that of commercial DAS-ELISA, and also could detect PVX and PVS in some samples that DAS-ELISA failed to detect. This multiplex RT-PCR technique demonstrates a higher sensitivity of virus detection than DAS-ELISA.Oligonucleotide microarray. This method was designed for simultaneous detection of seven potato viruses and one viroid. Recently, DNA microarray technique has been applied to detection of potato viruses. Use of reverse transcription labeling method made these DNA microarray systems had lower sensitivities, which was the major drawback of these systems. Hence, we attempted to establish an oligonucleotide microarray of high sensitivity and specificity for simultaneous detection of seven potato viruses and potato spindle tuber viroid （PSTVd）, using multiplex labeling PCR method and about 25mer oligonucleotides. Results showed that absence of cross hybridization occurred between labeled DNAs and heterogeneous probes, indicating that the oligonucleotide microarray established in this study was of high specificity. Through optimization of two multiplex PCR reactions, PLRV, PVY, PVX, PVA, PVS, CMV, TMV, PSTVd, and 18S rRNA could be simultaneously labeled. Combined with the multiplex labeling PCR method, the oligonucleotide microarray could detect CMV in the 10⁹ fold dilution of CMV RNA; however, the detection limit of nucleic acid spot hybridization technique was 10⁵ or 10⁶ fold dilution. Obviously, use of multiplex labeling PCR method significantly increased detection sensitivity of DNA microarray. The sensitivity of the oligonucleotide DNA microarray presented in this study was much higher than those of other DNA microarrays of detecting potato viruses reported by other researchers.Glass slide hybridization. This method was designed for detecting potato viruses or viroid from large numbers of potato samples in one reaction. As a diagnostic tool, DNA microarray has unique merit of simultaneous detection of multiple pathogens in one reaction. However, this technique is not competent for detecting few viruses in large numbers of potato samples. To address this problem, an attempt was made to establish a set of modified dot blot hybridization system, by replacement of ³²P-labeled probes with CY5-labeled probes and nylon membrane with aminosilane-coated glass slide as a RNA supporter. Results from conditional assays showed that formamide and DMSO spotting solutions used for nylon membrane were not suitable for aminosilane-coated glass slide; RNA dissolved in the two solutions showed lower binding efficiency than RNA dissolved in SSC solution. SSC （5×） of high concentration was beneficial to RNA binding on surface of aminosilane-coated glass slides; however, relatively low concentration （1×） showed obvious linear relativity. Various types of glass slides had different capabilities of binding RNA each other. Aminosilane-coated glass slides produced much higher hybridization signals than poly-L-lysine-coated slides and aldehyde-coated slides. Hence, sensitivity of the glass slide hybridization was determined using 1×SSC as spotting solution and aminosilane-coated glass slides as a RNA supporter. Probes prepared by PCR method and random labeling method could detect as little as 1.71 pg and 13.67 pg TMV RNA, respectively; nylon membrane hybridization with ³²P-labeled probe could detect 6.83 pg TMV RNA. Although faint cross-hybridization occurred between PSTVd-specific probe and total RNA sample isolated from PVY-infected Nicotiana glutinosa leaf tissue, absence of cross-hybridization aroused among three Potyvirus species, including PVA, PVY, and Zucchini yellow mosaic virus （ZYMV）, demonstrating that the established method possessed high specificity. A test of field-grown potato samples suggested that the method was capable of detecting potato virus（es） in a great number of potato samples.Every diagnostic method has its advantages and disadvantages, including the four sets of methods established in this study. For example, it will increase expense that use multiplex RT-PCR to detect single virus; use of VDA-RT-PCR to do will save expense （especial reagents for extraction of nucleic acid）, time and labor. If few viruses will be detected in large numbers of potato samples, we should choose glass slide nucleic acid hybridization, instead of oligonucleotide microarray method. If many viruses will be detected in few samples, we should select multiplex RT-PCR or oligonucleotide microarray, rather than glass slide nucleic acid hybridization. Therefore, we can pertinently select suitable diagnostic methods to decrease expense, time and labor as little as possible, according to different detecting requirements.更多还原