JIA Hui,ZHENG Jie,CAO Zhiyan,et al.Optimization of Hybridization Condition for Lily Virus Detection Microarray[J].Northern Horticulture,2015,39(21):110-115.[doi:10.11937/bfyy.201521029]
百合病毒检测芯片的杂交条件优化
- Title:
- Optimization of Hybridization Condition for Lily Virus Detection Microarray
- 文献标志码:
- A
- 摘要:
- 分别以感染百合无症病毒、黄瓜花叶病毒和百合斑驳病毒的百合及感染烟草花叶病毒和马铃薯病毒Y的烟草为试材，根据5种植物病毒外壳蛋白基因保守序列设计引物和寡核苷酸探针，并制备基因芯片。用Trizol 试剂盒提取感染病毒的植物总RNA，荧光RT-PCR产物与芯片杂交，研究PCR产物是否进行变性处理、杂交时间、杂交温度、杂交液组分SSC和SDS浓度及PCR体系中非荧光引物和荧光引物比例对芯片杂交的影响。结果表明：杂交适宜条件为6×SSC、0.2％ SDS的杂交液、42℃杂交60 min，PCR体系中非荧光与荧光引物比例为1∶10，PCR产物要进行变性处理。经过整体条件优化后的基因芯片在杂交检测上具有较高的特异性，适于检测百合病毒病。
- Abstract:
- In the present study the lily infected by Lily symptomless virus（LSV），Cucumber mosaic virus(CMV)，Lily mottle virus(LMoV) and tobacco infected by Tobacco mosaic virus (TMV)，Potato virus Y（PVY) were chosen as test material.Based on conserved coat protein region nucleotide sequences of five kinds of viruses，primers and probes were designed and the microarray were designed and fabricated.Total RNA of infected plant was extracted by the Trizol regent，and products that Cy3 labeled RT-PCR amplification hydrid with the microarray.The effects of whether the PCR products were denatured，hybridization time，hybridization temperature，hybridization buffer and the ratio of non-market primer to market primer with fluorescence in PCR system on microarray hybridization were evaluated.The result showed that the optimal hybridization time was 60 minutes；temperature was 42℃；SSC concentration was 6×SSC，SDS was 0.2%；The ratio of non-market primer to market primer with fluorescence was 1∶10 in the PCR system，and the PCR products must to be degenerated before hybridization.After the overall optimization，the gene chips had a high specificity in the hybridization，and could be used for detection of lily viruses fast and accurately.
参考文献/References:
[1]RYU K H，PARK H W，CHOI J K.Characterization and sequence analysis of lily isolate of cucumber mosaic virus from lilium tsingtauense[J].The Plant Pathology Journal，2002，18(2)：85-92. [2]ZHENG H Y，CHEN J，ZHAO M F，et al.Occurrence and sequences of Lily mottle virus and Lily symptomless virus in plants grown from imported bulbs in Zhejiang province[J].Archives of Virology，2003，148：2419-2428. [3]XU P S，LI H A，LIU J W，et al.Sequence and structure prediction of RNA-dependent RNA polymerase of Lily symptomless virus isolated from L.x ‘Casablanca’ [J].Archives of Virology，2011，156(6)：939-943. [4]ASJESC J，BLOM BARHOORN G J，PIRON P G M，et al.Control review of air-borne tulip breaking virus and Lily symptomless virus in Lilium in the Netherlands[J].Acta Horticulture，1996，432:290-297. [5]HAN L J，LIU W H.Studies on Prunus Necrotic Ringspot Virus (PNRSV) occurring on lily[J].Agricultural Sciences in China,2007，6(10):1201-1208. [6]CHINESTRA S C，FACCHINETTI C，CURVETTO N R,et al.Detection and frequency of Lily viruses in Argentina[J].Plant Disease，2019，94(10)：1189-1194. [7]BELLARDI M G，NANNI G，BERTACCINI A，et al.Old and new viruses of lily in Italy[J].Acta Horticulturae，2002，568:215-220. [8]宁红，秦蓁.分子生物学技术在检疫性有害生物诊断中的应用[J]．植物检疫，2002，16（2）：98-100. [9]邹立扣，王红宁.基因芯片技术检测细菌耐药性的研究进展[J]．微生物学杂志，2003，23(1)：30-32. [10]徐根明，丁先锋，朱聪，等.植物病毒检测芯片的杂交条件优化[J].中国生物工程杂志，2007，27(10)：75-80. [11]马淑华，王升启.cDNA芯片技术杂交效率的优化[J].药学学报，2002，37(2)：153-157. [12]DAWSON E D，REPPERT A E，ROWLEN K L，et al.Spotting optimization for oligo microarrays on aldehyde-glass[J].Analytical Biochemistry，2005，341(2)：352-360. [13]SHI G，WEN S Y，CHEN S H，et al Fabrication and optimization of the multiplex PCR based oligonucleotide microarray for detection of neisseria gonorrhoeae Chlamydia trachomatis and ureaplasma urealyticum[J].Journal of Microbiological Methods，2005，62(2)：245-256. [14]李长绿,王秀利,李宁,等.cDNA芯片制备条件的优化[J].农业生物技术学报，2005，13(4)：516-523. [15]温颖，马文丽，吴清华，等.60 mer长寡核苷酸微阵列构建的优化[J].广东医学，2005,26(4)：460-462. [16]贾慧，王艳辉，王进忠，等.基因芯片技术检测黄瓜花叶病毒、烟草花叶病毒和马铃薯Y病毒[J].华北农学报，2011，26(1)：83-86. [17]SHENG-CE T.Biochip-base devices and methods in microbial community ribotyping[J].Encyclopedia Environment Microbiol,2001(4):443-449. [18]YAN L，CHENG J.Detection of known mutation in hypertrphic cardiomyopathy using oligonucleotide microarrays assisted by improved base stacking hybridization[J].Biotechnology Letters,2003，25(6):1613-1618. [19]CHENG J，WANG D.Single nucleotide polymorphism discrimination assisted by improved base stacking hybridization using oligonucleotide microarrays[J].Bio Techniques，2003,35(8):300-308.
备注/Memo
第一作者简介：贾慧（1977-)，女，河北沽源人，硕士，讲师，现主要从事植物分子病理学等研究工作。E-mail：hui_jiahui@126.com.责任作者：董金皋（1963-)，男，河北平乡人，博士，教授，博士生导师，现主要从事植物真菌毒素研究及植物分子病理学等研究工作。基金项目：北京市自然科学基金资助项目（5043026）。