黄瓜细菌性角斑病PMA-qPCR检测方法的建立和应用

doi:10.13560/j.cnki.biotech.bull.1985.2016.02.009

摘要/Abstract

摘要： 黄瓜细菌性角斑病为黄瓜常见的一种细菌病害，其致病菌为丁香假单胞菌黄瓜致病型。目前该病在全球范围内已经造成严重的经济损失。针对于PMA染料能够区分细胞死活的特性将其与荧光定量PCR技术结合。(1)通过比对分析GenBank中该菌种不同株的gap1基因序列，找出gap1基因的保守区并根据保守区基因序列设计了一对特异性引物Dxf1和Dxr1，以其他5种非丁香假单胞菌基因组为模板进行实时定量PCR扩增，只有丁香假单胞菌有扩增曲线，证明引物非常特异。(2)以gap1基因为目的基因构建其克隆载体，将克隆载体导入到大肠杆菌感受态细胞中进行培养，使其大量复制。再将质粒提取，以提取的质粒作为标准品，根据其浓度将其稀释为5×10¹-5×10⁷7个梯度绘制标准曲线，获得的扩增效率为96.6%，并做组内重复和组间重复，变异系数均在2%内，证明标准曲线重复性良好。(3)将构建好的方法用于实际样品的检测，得到的Ct值为27.99，根据标准曲线所得的起始模板与Ct值之间的线性关系公式，检测到100 mg患病叶片中含有的菌体为7.69×10²拷贝。研究表明，该方法可以快速鉴定并检测实际样品中的活的致病菌的数量，为黄瓜细菌性角斑病的防控提供技术支持。

关键词: 黄瓜细菌性角斑病, PMA-qPCR, 丁香假单胞菌, 检测, 有活力菌体

Abstract: Bacterial angular leaf spot on cucumber is a common bacterial disease for cucumber, and its causative pathogen is Pseudomonas syringae pv.lachrymans.Currently this disease has caused a worldwide serious economic loss. Duo to the feature of PMA dyes that may distinguish whether the bacterial cells are alive or dead, PMA-qPCR was established by combining it with fluorescence quantitative PCR(qPCR)technology.(1)We compared the sequences of gene gap1 in different strains of the bacteria in GenBank, and found out the conservative area of gene gap1, and designed a pair of specific primers Dxf1 and Dxr1 for this bacterium according to the sequences of conservative area of gene gap1.Then we applied the qPCR method to do amplification using the genome of 5 non-Pseudomonas syringae pv.Lachrymans as template, and only P. syringae pv.lachrymans showed the positive result, proving that primers had specificity.(2)We used the gene gap1 as the target gene to construct a clone vector that was inserted into the competent cells of Escherichia coli.Then the E.coli cells were cultured to have a lot of copies.Using the extracted plasmids as the standard sample, and it was diluted to seven gradients of 5×10¹-5×10⁷ and then drew a standard curve.The amplification efficiency obtained from the standard curve was 96.6%, and the coefficients of variation both within groups and between groups were less than 2%, showing a fine repeatability of the standard curve.(3)We applied the PMA-qPCR method to detect the actual samples and gained a Ct value of 27.99.Based the linear correlation between initial template from the standard curve and Ct values, we measured 7.69×10² copies viable cells per 100 mg sick leaves.This study showed that PMA-qPCR method could quickly identify and quantify the number of living bacteria in actual samples, and provided a technical support for the prevention and control of bacterial angular leaf spot on cucumber.

Key words: bacterial angular leaf spot on cucumber, PMA-qPCR, Pseudomonas syringae, detection, viable cell

孔维文, 李云龙, 王敬琦, 刘婷婷, 陈南, 金一, 何晓青. 黄瓜细菌性角斑病PMA-qPCR检测方法的建立和应用[J]. 生物技术通报, 2016, 32(2): 70-75.

KONG Wei-wen, LI Yun-long, WANG Jing-qi, LIU Ting-ting, CHEN Nan, JIN Yi, HE Xiao-qing. The Establishment of PMA-qPCR for Detecting Bacterial Angular Leaf Spot on Cucumber and Its Preliminary Application[J]. Biotechnology Bulletin, 2016, 32(2): 70-75.

参考文献

[1]Carsner E.Angular-leafspot of cucumber：dissemination, overwintering, and control：Cooperative investigations between the University of Wisconsin and the Bureau of Plant Industry, United States Department of Agriculture[M].Government Printing Office, 1918.
[2]Dessert J, Baker L, Fobes J.Inheritance of reaction to Pseudomonas lachrymans in pickling cucumber[J].Euphytica, 1982, 31(3)：847-855.
[3]Kennedy BW, Alcorn S.Estimates of US crop losses to procaryote plant pathogens[J].Plant Disease, 1980, 64(7)：674-676.
[4]Watanabe Y, Ohuchi A.Angular leaf spot of cucumber in Japan[J].JARQ-Japan Agricultural Research Quarterly, 1983, 17(2)：112-119.
[5]Olczak-Woltman H, Schollenberger M, M?dry W, et al.Evaluation of cucumber(Cucumis sativus)cultivars grown in Eastern Europe and progress in breeding for resistance to angular leaf spot(Pseud-omonas syringae pv.lachrymans)[J].European Journal of Plant Pathology, 2008, 122(3)：385-393.
[6]Zhao XL, Sun JD, Cheng HT.Isolation of antagonistic bacteria ageinst angular leaf spot disease on cucumber[J].Journal-Shenyang Agricultural University, 2005, 36(3)：349.
[7] 孙福在, 何礼远.黄瓜细菌性角斑病病原菌与寄主范围鉴定[J].植物病理学报, 1988, 18(1)：23-28.
[8] Fogliano V, Gallo M, Vinale F, et al.Immunological detection of syringopeptins produced by Pseudomonas syringae pv.lachrymans[J].Physiol Mol Plant Pathol, 1999, 55(5)：255-261.
[9]陈爱华, 杨坚.酶联免疫吸附(ELISA)法在食品微生物检测中的应用[J].中国食品添加剂, 2004, 4：109-111.
[10]Olczak-Woltman H, Masny A, Bartoszewski G, et al.Genetic diversity of Pseudomonas syringae pv.lachrymans strains isolated from cucumber leaves collected in Poland[J].Plant Pathology, 2007, 56(3)：373-382.
[11]陈旭, 齐凤坤, 康立功, 等.实时荧光定量 PCR 技术研究进展及其应用[J].东北农业大学学报, 2010, (8)：148-155.
[12] 付春华, 陈孝平, 余龙江.实时荧光定量 PCR 的应用和进展[J].激光生物学报, 2006, 14(6)：466-471.
[13] Josephson K, Gerba C, Pepper I.Polymerase chain reaction detection of nonviable bacterial pathogens[J].Applied and Environmental Microbiology, 1993, 59(10)：3513-3515.
[14] Nocker A, Cheung CY, Camper AK.Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs.dead bacteria by selective removal of DNA from dead cells[J].Journal of Microbiological Methods, 2006, 67(2)：310-320.
[15] Nocker A, Sossa-Fernandez P, Burr MD, et al.Use of propidium monoazide for live/dead distinction in microbial ecology[J].Appl Environ Microbiol, 2007, 73(16)：5111-5117.
[16] 张振家, 郁继华, 王喜林.黄瓜细菌性角斑病防治试验初报[J].甘肃农业大学学报, 1989, 4：63-66.
[17]仝铁铮, 吴舒旭, 李丹, 等.基于 PMA-定量 PCR 选择性检测技术的病原菌消毒特性研究[J].环境科学, 2011, 32(4)：1120-1126.
[18]李聪聪, 余以刚, 邱杨, 等.PMA-qPCR 方法用于监测超声波灭菌效率的适用性及其应用[J].现代食品科技, 2012, 28(4)：409-411.
[19]Wang LJ, Tan QL, Zhu J, et al.Development of PMA-qPCR assay for rapid and accurate detection of viable Lactobacillus para-casei in fermented dairy products[J].Chinese Journal of Microecology, 2013, 25(1)：1-4.
[20]Matsenko N, Rijikova V, Kovalenko S.Comparison of SYBR Green I and TaqMan real-time PCR formats for the analysis of her2 gene dose in human breast tumors[J].Bulletin of Experimental Biology and Medicine, 2008, 145(2)：240-244.
[21]Zhe W, Qing C, Qian T, et al.Using PCR for rapid detection of Pseudomonas syringae pv.lachrymans[J].Plant Quarantine, 2011, 6：012.
[22] 王平, 樊金娟, 刘长远, 等.黄瓜细菌性角斑病的分子检测[J].中国农学通报, 2012, 28(25)：150-153.
[23]Fujimoto J, Watanabe K.Quantitative detection of viable bifidobacterium bifidum BF-1 cells in human feces by using propidium monoazide and strain-specific primers[J].Applied and Environmental Microbiology, 2013, 79(7)：2182-2188.
[24]Van Frankenhuyzen JK, Trevors JT, Flemming CA, et al.Optimization, validation, and application of a real-time PCR protocol for quantification of viable bacterial cells in municipal sewage sludge and biosolids using reporter genes and Escherichia coli[J].J Ind Microbiol Biotechnol, 2013, 4011：1251-1261.
[25]Salam KW, El-Fadel M, Barbour EK, et al.A propidium monoazide-quantitative PCR method for the detection and quantification of viable Enterococcus faecalis in large-volume samples of marine waters[J].Appl Microbiol Biotechnol, 2014, 9820：8707-8718.
[26]Kim S, Ko G.Using propidium monoazide to distinguish between viable and nonviable bacteria, MS2 and murine norovirus[J].Letters in Applied Microbiology, 2012, 55(3)：182-188.
[27]孙艳秋, 赵奎华, 曹远银, 等.黄瓜细菌性角斑病免疫胶体金检测试纸条的研制[J].植物病理学报, 2011, 412：131-138.
[28]孙艳秋, 刘长远, 曹远银, 等.黄瓜细菌性白枯病病菌检测试纸条的研制[J].中国植保导刊, 2014, 34(10)：8-11.
[29] Dinu LD, Bach S.Detection of viable but non-culturable Escherichia coli O157：H7 from vegetable samples using quantitative PCR with propidium monoazide and immunological assays[J].Food Control, 2013, 31(2)：268-273.