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Total RNA in tulips was extracted by Trizol method. Primers were designed according to the sequences of Tobacco rattle virus and 18S rRNA gene of plant. The corresponding sections were amplified by RT-PCR and the PCR products were labeled by Cy3-dCTP. The probes of plant virus, 18S rRNA gene and comparisons were designed and immobilized on chips. Labeled PCR products were hybridized with the probes and the signals were scanned by scanner and analyzed by GenePix Pro 4.0 software. Tobacco rattle virus was detected from tulips which were imported from Holand. The accuracy and sensitivity of the plant virus gene chip were proved. 相似文献
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应用细菌磁颗粒实时荧光RT-PCR检测南瓜花叶病毒 总被引：1，自引：0，他引：1
A novel real-time RT-PCR method, BMPs based real-time RT-PCR which integrated with magne-tic separation technique of bacterial magnetic particles(BMPs), was set up for detection of Squash mosaic virus(SqMV).After SqMV particles in crude sap were concentrated by BMPs, viral RNAs were released and detected by real time RT-PCR.The results indicated that BMPs based real-time RT-PCR was efficient, and the detection sensibility was equivalent to that of the Trizol based real-time RT-PCR, of which Trizol reagent was used for viral RNAs extration.Comparing to Trizol-based method, the BMPs-based method had advantages of simplicity on operation, time saving for RNA extraction and without using noxious organic chemicals. 相似文献
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南方水稻黑条矮缩病毒一步双重RT-PCR 检测技术及其应用 总被引：5，自引：0，他引：5
An one-step dual RT-PCR method was developed for Southern rice black-streaked dwarf virus (SRBSDV) detection from host plants and insect vector white-backed planthopper (Sogatella furcifera Horvath,Hemiptera: Delphacidae), from which two cDNA fragments of the viral genome S5 and S10 were amplified
simultaneously. Two primer pairs, S5-F1 / S5-R2 (5′-ttacaactggagaagcattaacacg-3′ / 5′-atgaggtattgcgtaactgagcc -3′) and S10-oF / S10-oR(5cgcgtcatctcaaactacag -3′ / 5′-tttgtcagcatctaaagcgc -3′), were selectedfrom 40 primer pairs based on SRBSDV genome sequences, and amplified viral S5 ORF1 fragment (819 bp) and cp gene fragment ( 682 bp), respectively. Using total RNA extracts from infected plant leaf tissue or individual planthopper adult as templates, one step RT-PCR reaction in the conditions of annealing temperature of 53℃ with the final concentrations of 240 nmol / L S5-F1 / S5-R2 and 120 nmol / L S10-oF / S10-oR generated a similar yield of two amplicons in argrose electrophoresis analysis. Sequence analysis confirmed the correct
result of the amplification. Additionally, several commercal RNA extraction kits was proven to be fit for the template preparation, and the protocol for total RNA extraction from plant leaf tissue and individual planthopper was simplified by sample grinding direct in eppendorf tube. This method can be used for SRBSDV detection of dried or 70% alcochol soaked planthopper samples stored over one month in room temprature. Key words: Southern rice black-streaked dwarf virus; Sogatella furcifera Horvath 相似文献
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进境豇豆种子携带种传病毒的检测与鉴定 总被引：2，自引：0，他引：2
Imported cowpea seeds were detected with growing test, ELISA assay and RT-PCR method. The ELISA results showed that cowpea seedlings with symptoms reacted positively with antibody against Southern bean mosaic virus (SBMV). The 979 bp of fragment could be amplified from two positive ELISA samples using primers specific for Southern cowpea mosaic virus (SCPMV), and the sequence determination results proved that the pathogen existing in imported cowpea seeds was SCPMV. The positive ELISA results with SBMV antibody could be further confirmed by RT-PCR amplification with specific primers designed to amplify the coat protein gene and 3' noncoding region of SCPMV and SBMV. The RT-PCR method presented here was suitable for molecular identification of SBMV and SCPMV in entry-exit plant quarantine laboratories. 相似文献
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A species-specific PCR assay was established for rapid and accurate detection of the oomycete pathogen Phytophthora tentaculata in diseased plant tissues and infected soil.A pair of species-specific primers Pt1/Pt2 were designed on the basis of Ras-related protein(Ypt1) gene sequences of the Phytophthora species.PCR amplification with the Pt primers resulted in a 386 bp product only from isolates of P.tentaculata.The detection threshold with Pt primers was 100 pg of genomic DNA.A nested PCR procedure was developed using Ypt1F/Ypt1R as the first-round amplification primers and Pt1/Pt2 as the second-round primers,which increased the detection sensitivity 100-fold to 1 pg.PCR using these Pt primers can also be used to detect P.tentaculata in naturally infected plant tissues and soil.The PCR-based method developed in this study provides a rapid and sensitive tool for detection of P.tentaculata. 相似文献
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RNAi with natural defence mechanism of homologous RNA degradation is widely used in research of antiviral plant. It is important to construct a highly efficent RNAi vector for transgenic plants of virus resis-tance. In this study, part fragments of coat protein gene of Potato virus Y (PVY) (451-750 bp) were inserted into the two expression vectors. Vector pROKY300 without intron and pHelY300 with PDK and CAT introns on the hpRNA stem were constructed. The silence efficiency of virus resistance of the two vectors was investigated as 88% (22/25)for pROKY300 and 92% (23/25) for pHelY300 through transient expression mediated by agroinfiltration. The results showed that both vectors were highly antiviral and elucidated the validity of RNAi-medicates resistance to virus. 相似文献
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广东番茄上检测到Tospovirus病毒 总被引：1，自引：0，他引：1
Some tomato samples possibly infected by tospovirus in Guangdong were detected with indirect ELISA and RT-PCR. The results showed that the virus infected tomato did not react with the antiserum of Tomato spotted wilt virus (TSWV), but about 500 bp fragment of RT-PCR shared 83%-84% nucleotide identities with N gene of those reported tospoviruses. The phylogenetic tree of the N gene fragment compared with those of other tospoviruses indicated that the virus infected tomato was belonged to Tospovirus. 相似文献
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葫芦种子传黄瓜绿斑驳花叶病毒的检测* 总被引：1，自引：0，他引：1
从感染黄瓜绿斑驳花叶病毒（Cucumber green mottle mosaic virus, CGMMV）的葫芦植株上收取种子,通过苗期症状观察法、双抗体夹心酶联免疫吸附法（DAS-ELISA）、免疫捕获反转录PCR（IC-RT-PCR）法测定葫芦种子的带毒情况,并用生物学接种方法测定葫芦种子携带病毒的侵染活性。苗期症状观察法结果表明,199株幼苗有2株表现花叶斑驳症状,种子传毒率为1.01%;而利用DAS-ELISA和IC-RT-PCR法随机检测30粒葫芦病株种子,CGMMV检出率为100%。种子各部位携带的CGMMV接种葫芦表现典型的花叶斑驳症状,表明葫芦种子携带的CGMMV具有侵染活性。DAS-ELISA检测葫芦种子CGMMV的灵敏度为1/5120种子研磨液。 相似文献
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From October 2017 to April 2019, a total of 290 suspected virus-infected watermelon leaf samples were collected in 7 urban areas (Wenchang, Wanning, Lingshui, Sanya, Ledong, Dongfang, Changjiang) in Hainan island. Nine viruses were detected including MYSV, WSMoV, CCYV, CMV, TMV, CGMMV, ZYMV, WMV and PRSV by small RNA deep sequencing and RT-PCR. The detection rates of MYSV, CCYV, CGMMV, ZYMV, CMV, WSMoV, PRSV, TMV and WMV were 66.90%, 25.86%, 22.07%, 17.59%, 10.00%, 7.59%, 5.52%, 2.76% and 1.03% respectively. The co-infection rate of virus was 56.56% and 14 types of mixed infections were detected. The results of this study clarify the identification and distribution of waterme-lon-infecting viruses in Hainan. 相似文献
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M. M. Mathioudakis V. I. Maliogka C. I. Dovas S. Paunovi&#; N. I. Katis 《Plant pathology》2009,58(2):228-236
In this study a spot nested RT-PCR assay was developed for the detection of Apple stem pitting virus (ASPV). A one step RT-PCR for the generic detection of foveaviruses using degenerate primers that target a conserved region of the RNA-dependent RNA polymerase (RdRp) gene was followed by a nested PCR that amplifies a 312 bp ASPV specific product. The method is rapid, simple and displays high sensitivity and broad detection range, overcoming the virus molecular variability. The optimum sampling conditions for reliable virus detection were also investigated. ASPV was detected throughout the year in different plant tissues of affected trees, thus the method could be used for routine screening and in certification schemes of pome fruits. ASPV was detected in quince orchards in Greece in all trees that were tested, showing a fruit deformation disorder. Sequencing and phylogenetic analysis of amplicons generated by RT-PCR from plant tissue affected with the deformation disease indicated that the agent responsible was a variant of ASPV. 相似文献
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随着大豆进口量的日益增加,大豆携带病毒传入我国的风险在不断增大,高效、快速地进行植物病毒检测,防止外来有害生物入侵,是目前各口岸植物检疫工作的重点。本文介绍了一种植物病毒检测的新方法—纳米上转换荧光技术。该技术是一种利用磁性纳米颗粒(magnetic nanoparticles, MNP)进行病毒分离、富集和定位;同时引入上转换荧光(up converting phosphor, UCP)材料作为标记物的免疫检测方法,具有高度的抗干扰性、多元性、稳定性和安全性等特点。 相似文献
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Seed disinfection treatments do not sufficiently eliminate the infectivity of Cucumber green mottle mosaic virus (CGMMV) on cucurbit seeds 
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下载免费PDF全文 Tobamoviruses induce crop diseases that are responsible for significant economic losses around the world. Like other tobamoviruses, Cucumber green mottle mosaic virus (CGMMV) forms highly stable particles that can persist for long periods on plant debris, in soil and on seed surfaces. These particles serve as a primary source of infection, infecting seedlings from which the virus can then be mechanically transmitted to other neighbouring plants. Contaminated seeds also provide a route for the movement of the virus between countries and its introduction into new areas. Effective seed disinfection treatments and the use of uncontaminated seed may reduce the global prevalence of this virus. Several treatments based on the use of heat or chemicals have been reported to effectively eliminate CGMMV and other tobamoviruses from seeds. An evaluation of these treatments on highly contaminated seed lots revealed inconsistent results, which encouraged the construction of a more accurate detection method that combines morphological, serological, molecular and biological analyses in one protocol. The detection of viable (infectious) viral particles in seed treated with heat, trisodium phosphate or a combined treatment, indicates that these treatments are insufficient. The serological detection of CGMMV in the inner parts of infected seeds provides a possible explanation for the inconsistent efficacy of these treatments. 相似文献
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侵染葫芦的黄瓜绿斑驳花叶病毒广西分离物分子鉴定 总被引：1，自引：0，他引：1
从广西南宁市郊温室大棚中的葫芦[Lagenaria siceraria(Molina)Stand.]上采集到一个表现脉绿、花叶症状的病毒样品,ELISA检测表明,该样品与黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)有密切的血清学关系,利用RT-PCR方法从样品中扩增获得约500bp的DNA片段,序列分析表明,该片段是CGMMV的外壳蛋白基因,暂将该病毒分离物定名为GX-BG。外壳蛋白基因核苷酸序列系统进化树分析表明,已报道的CGMMV主要分为3大群体,GX-BG与中国辽宁分离物(CGMMV-LN)分别属于不同的群体。 相似文献