茶尺蠖核型多角体病毒（EoNPV）实时荧光定量PCR检测方法的优化及应用

doi:10.13305/j.cnki.jts.2013.03.006

摘要/Abstract

摘要： 选用茶尺蠖核型多角体病毒（EoNPV）基因组中序列特异性很高的p16基因为目标基因,通过设计引物、PCR扩增、目的片段与载体连接转化获得含p16基因的重组质粒进行测序,进而以经测序鉴定的p16基因的PCR纯化产物作为实时荧光定量PCR标准品模板,稀释后建立SYBR Green I荧光定量标准曲线,统计分析显示标准品浓度的对数值与Ct值之间存在良好的线性关系（R²=0.9981）。该方法的检测灵敏度为10²,扩增产物形成单一的特异性熔解峰,组内组间变异系数均小于3%。同时分别对含有其他茶树害虫病毒和不同浓度的茶尺蠖病毒产品的样品进行检测,能明确样品中是否含有EoNPV及含量。根据已建立的方法对感染病毒后不同时间段的茶尺蠖幼虫进行检测,每克幼虫样本内p16基因拷贝数的增殖倍数对数值与感染时间呈正相关（R²=0.9935）,可以获得茶尺蠖幼虫感染病毒后不同时间的增殖动态。上述结果表明,该方法能定性定量鉴定茶尺蠖核型多角体病毒,可用于茶尺蠖核型多角体病毒类生物农药的检测和感染过程检测。

关键词: EoNPV, SYBR Green I, 定性定量, 检测

Abstract: Primers were designed based on p16 gene from Ectropis obliqua nucleopolyhedrovirus in order to optimize the detection method of SYBR Green I real-time fluorescence quantitation PCR for EoNPV, and the standard curve of SYBR Green I real-time fluorescence quantitative PCR for p16 gene was established using the recombinant plasmid DNA as template. Statistic analysis showed that there was a good linear relationship between Ct value and the logarithmic value of plasmid concentrations (R²=0.9981). The sensitivity of the method was 10² copies/μl, and wide detection range of 6 orders of magnitude was obtained. Larave infected at different time were sampled and detected by the method, and the results showed that there was a good linear relation between the logarithmic value of the multiples of gene copies and the infection time (R²=0.9935). Experimental results indicated that the method can identify biological pesticides from qualitative and quantitative aspects, and distinguish the high homology nuclearpolyhedrosisvirus (Euproctis pseudoconspersa nuclearpolyhedrosisvirus) from EoNPV accurately.

Key words: EoNPV, SYBR Green I, qualitative and quantitative, detection

中图分类号:

S435.711

袁志军, 张传溪, 肖强, 殷坤山. 茶尺蠖核型多角体病毒（EoNPV）实时荧光定量PCR检测方法的优化及应用[J]. 茶叶科学, 2013, 33(3): 229-236. doi: 10.13305/j.cnki.jts.2013.03.006.

YUAN Zhi-jun, ZHANG Chuan-xi, XIAO Qiang, YIN Kun-shan. Optimization and Application of Real-time Fluorescence Quantitative PCR for Detection of Ectropis obliqua Nucleopolyhedrovirus[J]. Journal of Tea Science, 2013, 33(3): 229-236. doi: 10.13305/j.cnki.jts.2013.03.006.

参考文献 29

[1]	Barrois M, Bieche I, Mazoyer S, et al. Real-time PCR-based gene dosage assay for detecting BRCA1 rearrangements in breast-ovarian cancer families[J]. Clin Genet, 2004, 65(2): 131-136.
[2]	Baskin D G, Bastian L S.Immuno-laser capture microdissection of rat brain neurons for real time quantitative PCR[J]. Methods Mol Biol, 2010, 588: 219-230.
[3]	Batista-Dos-Santos S, Raiol M, Santos S, et al. Real-time PCR diagnosis of Plasmodium vivax among blood donors[J]. Malar J, 2012, 11(1): 345.
[4]	Bauer M, Patzelt D.Identification of menstrual blood by real time RT-PCR: technical improvements and the practical value of negative test results[J]. Forensic Sci Int, 2008, 174(1): 55-59.
[5]	Becker S, Franco J R, Simarro PP, et al. Real-time PCR for detection of Trypanosoma brucei in human blood samples[J]. Diagn Microbiol Infect Dis, 2004, 50(3): 193-199.
[6]	Behets J, Declerck P, Delaedt Y, et al. A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples[J]. Water Res, 2007, 41(1): 118-126.
[7]	Bel Y, Ferre J, Baltassar E, et al. Quantitative real-time PCR with SYBR Green detection to assess gene duplication in insects: study of gene dosage in Drosophila melanogaster (Diptera) and in Ostrinia nubilalis (Lepidoptera)[J]. BMC Res Notes, 2011, 4: 84.
[8]	Bell A S, Blanford S, Jenkins N, et al. Real-time quantitative PCR for analysis of candidate fungal biopesticides against malaria: technique validation and first applications[J]. J Invertebr Pathol, 2009, 100(3): 160-168.
[9]	Bernard P S, Wittwer C T.Real-time PCR technology for cancer diagnostics[J]. Clin Chem, 2002, 48(8): 1178-1185.
[10]	Berrada H, Soriano J M, Pico Y, et al. Quantification of Listeria monocytogenes in salads by real time quantitative PCR[J]. Int J Food Microbiol, 2006, 107(2): 202-206.
[11]	Bidet P, Liguori S, Lauzanne De A, et al. Real-time PCR measurement of persistence of Bordetella pertussis DNA in nasopharyngeal secretions during antibiotic treatment of young children with pertussis[J]. J Clin Microbiol, 2008, 46(11): 3636-3638
[12]	姚勤, 高路, 陈克平, 等. 荧光定量PCR检测家蚕核型多角体病毒在其宿主体内的增殖动态[J]. 昆虫学报, 2005 (6): 871-875.
[13]	乔鲁芹, 曲良建, 王玉珠, 等. 美国白蛾核型多角体病毒实时荧光定量PCR检测方法的建立及应用[J]. 昆虫学报, 2010, 53(7): 824-830.
[14]	张益民, 王学兰, 张世敏. 茶尺蠖核型多角体病毒超微结构的初步研究[J]. 科学通报, 1985(24): 1918-1920.
[15]	殷坤山, 陈华才. 喷施茶尺蠖病毒杀虫剂对茶叶品质的影响[J]. 中国茶叶, 2002, 24(4): 5.
[16]	杜军利, 张传溪, 肖强, 等. 茶尺蠖核型多角体病毒荧光定量PCR检测方法的建立[J]. 茶叶科学, 2010, 30(3): 203-207.
[17]	Rutledge RG, Stewart D.Critical evaluation of methods used to determine amplification efficiency refutes the exponential character of real-time PCR[J]. BMC Molecular Biology, 2008, 9: 96.
[18]	Karlen Y, McNair A, Perseguers S, et al. Statistical significance of quantitative PCR[J/OL]. BMC Bioinformatics, 2007, 8: 131. doi: 10.1186/1471-2105-8-131.
[19]	Feng J, Zeng R, Chen J.Accurate and efficient data processing for quantitative real-time PCR using a tripartite plant virus as a model[J/OL]. Biotechniques, 2008, 44: 901-912. doi: 10.2144/000112750.
[20]	杨发龙, 岳华, 张焕容, 等. Real-time PCR在病毒学研究中的应用[J]. Heilongjiang Animal Science and Veterinary Medicine, 2008(3): 22-23.
[21]	王学波, 李建远. 人线粒体DNA荧光定量PCR 检测方法的建立[J]. 生物医学工程研究, 2008, 27(4): 298-301.
[22]	Swillens S, Dessars B, Housni HE.Revisiting the sigmoidal curve fitting applied to quantitative real-time PCR data[J/OL]. Anal Biochem, 2008, 373: 370-376. doi: 10.1016/j.ab.2007.10.019.
[23]	Tichopad A, Dilger M, Schwarz G, et al. Standardized determination of real-time PCR efficiency from a single reaction set-up[J/OL]. Nucleic Acids Res, 2003, 31: e122. doi: 10.1093/nar/gng122.
[24]	Schefe J H, Lehmann K E, Buschmann I R, et al. Quantitative real-time RT-PCR data analysis: current concepts and the novel ‘gene expression's CT difference’ formula[J]. Journal of Molecular Medicine, 2006, 84(11): 901-910.
[25]	Santhosh S R, Parida M M, Dash P K, et al. Development and evaluation of SYBR Green I-based one step real-time RT-PCR assay for detection and quantification of Chikungunya virus[J]. Journal of Clinical Virology, 2007, 39: 188-193.
[26]	殷坤山, 陈华才, 肖强, 等. 茶尺蠖核型多角体病毒制剂的试制与推广应用[J]. 中国病毒学, 2000, 15: 81-84.
[27]	冷杨, 肖强, 殷坤山. 茶毛虫核型多角体病毒Bt混剂的作用特性[J]. 植物保护学报, 2007, 34(2): 177-181.
[28]	Ozaki H, McLaughlin L W. The estimation of distances between specific backbone-labeled sites in DNA using fluorescence resonance energy transfer[J]. Nucleic Acids Res, 1992, 20(19): 5205-5214.
[29]	Mackay M, Arden KE, Nitsche A.Real-time fluorescent PCR techniques to study microbial-host interactions. Methods in Microbiology[J]. 2004, 34: 255-330.