生物技术通报 ›› 2021, Vol. 37 ›› Issue (7): 183-190.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0579
方圆1(), 吴迅1, 林宇2, 王海燕1, 吴慧平1, 鞠玉亮1()
收稿日期:
2021-04-30
出版日期:
2021-07-26
发布日期:
2021-08-13
作者简介:
方圆,男,硕士研究生,研究方向:植物线虫学;E-mail: 基金资助:
FANG Yuan1(), WU Xun1, LIN Yu2, WANG Hai-yan1, WU Hui-ping1, JU Yu-liang1()
Received:
2021-04-30
Published:
2021-07-26
Online:
2021-08-13
摘要:
旨在建立一种以重组酶聚合酶扩增技术(RPA)为基础的快速检测方法,用于松材线虫和拟松材线虫的同步检测鉴定。根据松材线虫和拟松材线虫ITS区差异序列,分别设计松材线虫和拟松材线虫特异性上游引物Bx-rpa-F、Bm-rpa-F和两者通用下游引物Bxm-rpa-R。优化3条引物在反应体系中的最佳配比,建立双重RPA检测体系,并对其特异性和灵敏度进行分析。研究结果显示,双重RPA在37℃恒温条件反应30 min,即可完成对松材线虫和拟松材线虫核酸的同步扩增。Bx-rpa-F/Bm-rpa-F/Bxm-rpa-R对松材线虫的扩增片段为346 bp,对拟松材线虫的扩增产物为189 bp,且三者配比为5∶3∶8时,双重RPA扩增效果最好。双重RPA对松材线虫和拟松材线虫具有较高的检测特异性,对松材线虫的检测极限为10 pg/μL,相当于1/100条线虫,对拟松材线虫的检测极限为100 pg/μL,相当于1/10条线虫,其灵敏度低于常规PCR技术,但其满足对单条线虫检测的需求。双重RPA从松木样品中同步检测到松材线虫和拟松材线虫,操作简便、检测效率高、特异性强、灵敏度高、对仪器设备要求低,为松材线虫的检疫鉴定提供新方法。
方圆, 吴迅, 林宇, 王海燕, 吴慧平, 鞠玉亮. 松材线虫和拟松材线虫双重RPA检测研究[J]. 生物技术通报, 2021, 37(7): 183-190.
FANG Yuan, WU Xun, LIN Yu, WANG Hai-yan, WU Hui-ping, JU Yu-liang. Duplex-RPA Detection for Bursaphelenchus xylophilus and Bursaphelenchus mucronatus[J]. Biotechnology Bulletin, 2021, 37(7): 183-190.
种类Species | 种群 Population | 来源 Origin |
---|---|---|
松材线虫B. xylophilus | Bxah1 | 中国安徽 Anhui,China |
Bxah2 | 中国安徽 Anhui,China | |
Bxah3 | 中国安徽 Anhui,China | |
Bxjs | 中国江苏 Jiangsu,China | |
Bxus | 美国 USA | |
拟松材线虫B. mucronatus | Bmah1 | 中国安徽 Anhui,China |
Bmah2 | 中国安徽 Anhui,China | |
Bmis | 中国江苏 Jiangsu,China | |
Bmsk | 韩国South Korea | |
豆伞滑刃线虫B. doui | Bdsk | 韩国 South Korea |
食菌伞滑刃线虫B. fungivous | Bfsk | 韩国 South Korea |
莱奴尔夫滑刃线虫B. rainulfi | Brsk | 韩国 South Korea |
菊花滑刃线虫A. ritzemabosi | Arus | 美国 USA |
水稻干尖线虫A. besseyi | Abah1 | 中国安徽 Anhui,China |
水稻干尖线虫A. besseyi | Abah12 | 中国安徽 Anhui,China |
表1 供试线虫种群
Table 1 Population information of nematodes
种类Species | 种群 Population | 来源 Origin |
---|---|---|
松材线虫B. xylophilus | Bxah1 | 中国安徽 Anhui,China |
Bxah2 | 中国安徽 Anhui,China | |
Bxah3 | 中国安徽 Anhui,China | |
Bxjs | 中国江苏 Jiangsu,China | |
Bxus | 美国 USA | |
拟松材线虫B. mucronatus | Bmah1 | 中国安徽 Anhui,China |
Bmah2 | 中国安徽 Anhui,China | |
Bmis | 中国江苏 Jiangsu,China | |
Bmsk | 韩国South Korea | |
豆伞滑刃线虫B. doui | Bdsk | 韩国 South Korea |
食菌伞滑刃线虫B. fungivous | Bfsk | 韩国 South Korea |
莱奴尔夫滑刃线虫B. rainulfi | Brsk | 韩国 South Korea |
菊花滑刃线虫A. ritzemabosi | Arus | 美国 USA |
水稻干尖线虫A. besseyi | Abah1 | 中国安徽 Anhui,China |
水稻干尖线虫A. besseyi | Abah12 | 中国安徽 Anhui,China |
引物 Primer | 序列 Sequence(5'-3') | 长度Length/bp | 扩增长度 Amplification length/bp |
---|---|---|---|
Bx-rpa-F | TTCGTGCTCGTCACGATGATGCGATTGGTGACT | 33 | 346 |
Bm-rpa-F | AAGTCTGGGTTTCTATGCGCTGCGTTGAGTCGA | 33 | 189 |
Bxm-rpa-R | CGCAATTCACTGCGTTCTTCATCGACCCGCGAG | 33 | -- |
表2 用于双重RPA的引物及其序列
Table 2 Primers and their sequences for duplex-RPA
引物 Primer | 序列 Sequence(5'-3') | 长度Length/bp | 扩增长度 Amplification length/bp |
---|---|---|---|
Bx-rpa-F | TTCGTGCTCGTCACGATGATGCGATTGGTGACT | 33 | 346 |
Bm-rpa-F | AAGTCTGGGTTTCTATGCGCTGCGTTGAGTCGA | 33 | 189 |
Bxm-rpa-R | CGCAATTCACTGCGTTCTTCATCGACCCGCGAG | 33 | -- |
编号 Code | 采集地点 Collecting location | 双重RPA检测结果 Results from duplex-RPA detection | |
---|---|---|---|
B. xylophilus | B. mucronatus | ||
AN01 | 安徽南陵 Nanling,Anhui | + | - |
AN02 | 安徽南陵 Nanling,Anhui | + | - |
AN03 | 安徽南陵 Nanling,Anhui | + | + |
AC01 | 安徽滁州 Chuzhou,Anhui | + | |
AC02 | 安徽滁州 Chuzhou,Anhui | + | + |
AC03 | 安徽滁州 Chuzhou,Anhui | + | + |
AT01 | 安徽桐城Tongcheng,Anhui | + | - |
AT02 | 安徽桐城 Tongcheng,Anhui | - | + |
AT03 | 安徽桐城 Tongcheng,Anhui | - | + |
表3 松木样本中松材线虫与拟松材线虫的双重RPA检测
Table 3 Duplex-RPA detection for B. xylophilus and B. mucronatus
编号 Code | 采集地点 Collecting location | 双重RPA检测结果 Results from duplex-RPA detection | |
---|---|---|---|
B. xylophilus | B. mucronatus | ||
AN01 | 安徽南陵 Nanling,Anhui | + | - |
AN02 | 安徽南陵 Nanling,Anhui | + | - |
AN03 | 安徽南陵 Nanling,Anhui | + | + |
AC01 | 安徽滁州 Chuzhou,Anhui | + | |
AC02 | 安徽滁州 Chuzhou,Anhui | + | + |
AC03 | 安徽滁州 Chuzhou,Anhui | + | + |
AT01 | 安徽桐城Tongcheng,Anhui | + | - |
AT02 | 安徽桐城 Tongcheng,Anhui | - | + |
AT03 | 安徽桐城 Tongcheng,Anhui | - | + |
图1 松材线虫和拟松材线虫双重RPA检测 1:松材线虫;2:拟松材线虫;3:松材线虫与拟松材线虫混合样本;4:阴性对照;M:DNA marker DL2000
Fig. 1 Duplex-RPA assay for detecting B. xylophilus and B. mucronatus 1:B. xylophilus. 2:B. mucronatus. 3:Mixed sample of B. xylophilus and B. mucronatus. 4:Negative control. M:DNA marker DL2000
图2 不同引物配比下的扩增产物 1-9:Bx-rpa-F、Bm-rpa-F和Bxm-rpa-R的添加量分别为2.4:0:2.4、2.1:0.3:2.4、1.8:0.6:2.4、1.5:0.9:2.4、1.2:1.2:2.4、0.9:1.5:2.4、0.6:1.8:2.4、0.3:2.1:2.4、0:2.4:2.4 μL;M:DNA marker DL2000
Fig. 2 Amplified products with different primer ratios 1-9:The contents of Bx-rpa-F,Bm-rpa-F and Bxm-rpa-R were 2.4:0:2.4,2.1:0.3:2.4,1.8:0.6:2.4,1.5:0.9:2.4,1.2:1.2:2.4,0.9:1.5:2.4,0.6:1.8:2.4,0.3:2.1:2.4,0:2.4:2.4 μL,respectively. M:DNA marker DL2000
图3 双重RPA特异性检测
Fig. 3 Specificity test of duplex-RPA 1:B. xylophilus Bxah1;2:B. xylophilus Bxah2;3:B. xylophilus Bxah3;4:B. xylophilus Bxjs;5:B. xylophilus Bxus;6:B. mucronatus Bmah1;7:B. mucronatus Bmah2;8:B. mucronatus Bmis;9:B. mucronatus Bmsk;10:B. doui Bdsk;11:B. fungivous Bfsk;12:B. rainulfi Brsk;13:A. ritzemabosi Arus;14:A. besseyi Abah1;15:A. besseyi Abah2
图4 双重RPA灵敏度检测 A:双重RPA与常规PCR对松材线虫的检测灵敏度;B:双重RPA与常规PCR对拟松材线虫的检测灵敏度;C:双重RPA对松材线虫与拟松材线虫DNA等比例混合样品的检测灵敏度。1-6:10-1、10-2、10-3、10-4、10-5、10-6条线虫;M:DNA marker DL2000
Fig. 4 Sensitivity test of duplex-RPA A:Sensitivity of duplex-RPA and conventional PCR in the detection of B. xylophilus. B:Sensitivity of duplex-RPA and conventional PCR in the detection of B. mucronatus. C:Sensitivity of duplex-RPA in the detection of B. xylophilus and B. mucronatus in equal proportion.1-6:10-1,10-2,10-3,10-4,10-5,and 10-6 of a single nematode.M:DNA marker DL2000
图5 松木样品中松材线虫和拟松材线虫双重RPA检测 1-9:AN01、AN02、AN03、AC01、AC02、AC03、AT01、AT02、AT03 RPA检测结果;10:阴性对照;M:DNA marker DL2000
Fig. 5 Duplex-RPA assay for detecting B. xylophilus and B. mucronatus from wood samples 1-9:RPA test results of AN01, AN02, AN03, AC01, AC02, AC03, AT01, AT02 and AT03;10:negative control;M:DNA marker DL2000
[1] |
Jones JT, Haegeman A, Danchin EG, et al. Top 10 plant-parasitic nematodes in molecular plant pathology[J]. Mol Plant Pathol, 2013, 14(9):946-961.
doi: 10.1111/mpp.12057 pmid: 23809086 |
[2] | Mota MM, Vieira P. Pine wilt disease:A worldwide threat to forest ecosystems[M]. Dordrecht:Springer Netherlands, 2008. |
[3] |
Robertson L, Cobacho Arcos S, Escuer M, et al. Incidence of the pinewood nematode Bursaphelenchus xylophlius Steiner & Buhrer, 1934(Nickle, 1970)in Spain[J]. Nematology, 2011, 13(6):755-757.
doi: 10.1163/138855411X578888 URL |
[4] |
Zhao L, Mota M, Vieira P, et al. Interspecific communication between pinewood nematode, its insect vector, and associated microbes[J]. Trends Parasitol, 2014, 30(6):299-308.
doi: 10.1016/j.pt.2014.04.007 URL |
[5] | 程瑚瑞, 林茂松, 黎伟强, 等. 南京黑松上发生的萎蔫线虫病[J]. 森林病虫通讯, 1983, 2(4):1-5. |
Cheng HR, Lin MS, Li WQ, et al. Pine wilt disease on Pinus thunbergia Parl. In Nanjing[J]. Forest Pest and Disease, 1983, 2(4):1-5. | |
[6] |
Sultana T, Han H, Park JK. Comparison of complete mitochondrial genomes of pine wilt nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus(Nematoda:Aphelenchoidea)and development of a molecular tool for species identification[J]. Gene, 2013, 520(1):39-46.
doi: 10.1016/j.gene.2013.02.006 pmid: 23434520 |
[7] | 薛美静, 李英, 张靖, 等. 拟松材线虫纤维素酶活性与其致病性关系[J]. 生物安全学报, 2019, 28(3):181-188. |
Xue MJ, Li Y, Zhang J, et al. Studies on correlation between cellulase activity of Bursaphelenchus mucronatus and its pathogenicity[J]. J Biosaf, 2019, 28(3):181-188. | |
[8] |
Akbulut S, Yüksel B, Serin M, et al. Comparison of pathogenic potential of Bursaphelenchus species on conifer seedlings between greenhouse and outdoor conditions[J]. Phytoparasitica, 2015, 43(2):209-214.
doi: 10.1007/s12600-014-0433-2 URL |
[9] |
Zhou LF, Chen FM, Wang JC, et al. Virulence of Bursaphelenchus mucronatus to pine seedlings and trees under field cownditions[J]. For Path, 2016, 46(6):643-651.
doi: 10.1111/efp.12285 URL |
[10] | 叶建仁. 松材线虫病在中国的流行现状、防治技术与对策分析[J]. 林业科学, 2019, 55(9):1-10. |
Ye JR. Epidemic status of pine wilt disease in China and its prevention and control techniques and counter measures[J]. Sci Silvae Sin, 2019, 55(9):1-10. | |
[11] | 俞春来, 何洁, 刘乐乐, 等. 松材线虫雌虫尾尖突变化综述[J]. 安徽农业科学, 2017, 45(27):168-171. |
Yu CL, He J, Liu LL, et al. Report of variation of Bursaphelenchus xylophilus female tail shape[J]. J Anhui Agric Sci, 2017, 45(27):168-171. | |
[12] |
Kikuchi T, Aikawa T, Oeda Y, et al. A rapid and precise diagnostic method for detecting the Pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification[J]. Phytopathology, 2009, 99(12):1365-1369.
doi: 10.1094/PHYTO-99-12-1365 pmid: 19900002 |
[13] |
Min YY, Toyota K, Sato E. A novel nematode diagnostic method using the direct quantification of major plant-parasitic nematodes in soil by real-time PCR[J]. Nematology, 2012, 14(3):265-276.
doi: 10.1163/156854111X601678 URL |
[14] | 赵立荣, 廖金铃, 钟国强. 松材线虫和拟松材线虫的PCR快速检测[J]. 华南农业大学学报, 2005, 26(2):59-61. |
Zhao LR, Liao JL, Zhong GQ. A rapid method to detect Bursaphelenchus xylophilus and B. mucronatus by PCR[J]. J South China Agric Univ, 2005, 26(2):59-61. | |
[15] | 陈凤毛, 叶建仁, 汤坚, 等. 应用PCR-RFLP技术鉴别松材线虫与拟松材线虫[J]. 南京林业大学学报:自然科学版, 2006, 30(4):5-9. |
Chen FM, Ye JR, Tang J, et al. Identification of Bursaphelenchus xylophilus and b. mucronatus by PCR-RFLP technique[J]. J Nanjing For Univ:Nat Sci Ed, 2006, 30(4):5-9. | |
[16] | 陈凤毛, 叶建仁, 吴小芹, 等. 松材线虫两种实用分子检测技术[J]. 北京林业大学学报, 2011, 33(4):149-152. |
Chen FM, Ye JR, Wu XQ, et al. Two kinds of applied molecular skills to detect Bursaphelenchus xylophilus[J]. J Beijing For Univ, 2011, 33(4):149-152. | |
[17] | Hu YQ, Kong XC, Wang XR, et al. Direct PCR-based method for detecting Bursaphelenchus xylophilus, the pine wood nematode in wood tissue of Pinus massoniana[J]. For Pathol, 2011, 41(2):165-168. |
[18] |
Ye W, Giblin-Davis RM. Molecular characterization and development of real-time PCR assay for pine-wood nematode Bursaphelenchus xylophilus(Nematoda:Parasitaphelenchidae)[J]. PLoS One, 2013, 8(11):e78804.
doi: 10.1371/journal.pone.0078804 URL |
[19] | 何旭峰, 彭焕, 丁中, 等. 植物罹病组织中象耳豆根结线虫的LAMP快速检测方法[J]. 中国农业科学, 2013, 46(3):534-544. |
He XF, Peng H, Ding Z, et al. Loop-mediated isothermal amplification assay for rapid diagnosis of Meloidogyne enterolobii directly from infected plants[J]. Sci Agric Sin, 2013, 46(3):534-544. | |
[20] | 魏洪岩, 王暄, 李红梅, 等. 采用环介导等温扩增法(LAMP)快速检测苹果根结线虫[J]. 植物保护学报, 2016, 43(2):260-266. |
Wei HY, Wang X, Li HM, et al. Loop-mediated isothermal amplification assay for rapid diagnosis of Meloidogyne Mali[J]. J Plant Prot, 2016, 43(2):260-266. | |
[21] | 白宗师, 秦萌, 赵立荣, 等. 水稻干尖线虫的环介导恒温扩增技术(LAMP)快速检测方法[J]. 中国水稻科学, 2017, 31(4):432-440. |
Bai ZS, Qin M, Zhao LR, et al. Loop-mediated isothermal amplification assay for rapid diagnosis of Aphelenchoides besseyi[J]. Chin J Rice Sci, 2017, 31(4):432-440. | |
[22] | 刘星彤, 林柏荣, 廖金铃, 等. 环介导等温扩增法(LAMP)检测最短尾短体线虫[J]. 华中农业大学学报, 2018, 37(6):17-24. |
Liu XT, Lin BR, Liao JL, et al. Detection of Pratylenchus brachyurus by loop-mediated isothermal amplification[J]. J Huazhong Agric Univ, 2018, 37(6):17-24. | |
[23] |
Subbotin SA. Recombinase polymerase amplification assay for rapid detection of the root-knot nematode Meloidogyne enterolobii[J]. Nematology, 2019, 21(3):243-251.
doi: 10.1163/15685411-00003210 |
[24] |
Cha D, Kim D, Choi W, et al. Point-of-care diagnostic(POCD)method for detecting Bursaphelenchus xylophilus in pinewood using recombinase polymerase amplification(RPA)with the portable optical isothermal device(POID)[J]. PLoS One, 2020, 15(1):e0227476.
doi: 10.1371/journal.pone.0227476 URL |
[25] |
Ju YL, Lin Y, Yang GG, et al. Development of recombinase polymerase amplification assay for rapid detection of Meloidogyne incognita, M. javanica, M. Arenaria, and M. enterolobii[J]. Eur J Plant Pathol, 2019, 155(4):1155-1163.
doi: 10.1007/s10658-019-01844-6 URL |
[26] |
Chi YK, Zhao W, Ye MD, et al. Evaluation of recombinase polymerase amplification assay for detecting Meloidogyne javanica[J]. Plant Dis, 2020, 104(3):801-807.
doi: 10.1094/PDIS-07-19-1473-RE URL |
[27] | 林宇, 鞠玉亮, 刘娟, 等. 菊花滑刃线虫RPA检测方法研究[J]. 湖南农业科学, 2021(1):1-3, 10. |
Lin Y, Ju YL, Liu J, et al. Development of RPA assay for detection of Aphelenchoides ritzemabosi[J]. Hunan Agric Sci, 2021(1):1-3, 10. | |
[28] | 容万韬, 王金成, 刘鹏, 等. 单条线虫DNA提取方法研究[J]. 华北农学报, 2014, 29(2):127-132. |
Rong WT, Wang JC, Liu P, et al. Research on DNA extraction methods from a single nematode[J]. Acta Agric Boreali Sin, 2014, 29(2):127-132. | |
[29] | 于海英, 吴昊. 辽宁发现松材线虫新寄主植物和新传播媒介昆虫[J]. 中国森林病虫, 2018, 37(5):61. |
Yu HY, Wu H. Discovery of new host plant and new vector insects of Bursaphelenchus xylophilus in Liaoning Provence[J]. For Pest Dis, 2018, 37(5):61. | |
[30] | 李娟, 姚翰文. 我国松材线虫病疫区现状及管理对策[J]. 中国森林病虫, 2019, 38(3):45-46. |
Li J, Yao HW. Current situation and management countermeasures of pine wilt disease in China[J]. For Pest Dis, 2019, 38(3):45-46. | |
[31] | 于海英, 吴昊, 张旭东, 等. 落叶松自然条件下感染松材线虫初报[J]. 中国森林病虫, 2019, 38(4):7-10. |
Yu HY, Wu H, Zhang XD, et al. Preliminary study on Larix spp. infected by Bursaphelenchus xylophilus in natural environment[J]. For Pest Dis, 2019, 38(4):7-10. |
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