|
|
|
| Establishment and Optimization of Single-tube Nested PCR Detection Technique for Phytoplasma Related to Sisal Purple Leafroll Disease |
| LU Peng-Peng1,2, WU Wei-Huai2,*, ZHENG Jin-Long2, WANG Gui-Hua2, HE Chun-Ping2, LIN Pei-Qun2, HUANG Xing2, LIANG Yan-Qiong2, YI Ke-Xian2,* |
1 College of Plant Protection, Hainan University, Haikou 570228, China;
2 Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests/Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China |
|
|
|
|
Abstract Sisal purple leafroll disease (SPLD) is a devastating disease that has occurred on sisal (Agave sisalana) in recent years. Previous studies by this research group have shown that the disease is highly associated with phytoplasmas. It is necessary to establish an efficient molecular detection technology for in-depth research on phytoplasma or disease monitoring and detection. To this end, this study intends to establish an efficient single-tube nested PCR detection technique for phytoplasma related to sisal purple leafroll disease. Firstly, the annealing temperature of inner and outer primers in the single tube nested PCR reaction system was studied by single-factor test. After the annealing temperature was determined, an orthogonal design was used to optimize key factors such as inner and outer primer concentration, dNTPs concentration, and Ex Taq enzyme dosage of single-tube nest PCR detection system. The results showed that when the annealing temperature of outer primer Sis-F1/R1 was determined to be 64 °C, and inner primer Sis-F2/R2 was 54 °C, the best reaction system (25 μL) was finally selected by orthogonal design test as follows: 10×Ex Taq Buffer 2.5 μL, 2.5 mmol/L dNTPs 5 μL, Ex Taq DNA Polymerase 1 μL, 15 μmol/L inner primers Sis-F2/R2 each 1 μL, 0.02 μmol/L outer primers Sis-F1/R1 each 1 μL, template DNA 1 μL, ddH2O 11.5 μL. The above reaction system only amplified bands from the DNA template of the plants with purple leafroll disease, which had a high degree of specificity. The lowest detection limit of the detection system was a phytoplasma concentration of ≥ 1 fg/μL. The single tube nested PCR reaction system for the detection of phytoplasma related to sisal purple leafroll disease established in this research could provide technical support for follow-up researches on disease monitoring, correlation investigation, pathogenicity and functional verification.
|
|
Received: 30 November 2020
|
|
|
|
Corresponding Authors:
*weihuaiwu2002@163.com;yikexian@126.com
|
|
|
|
[1] 安凤秋, 吴云锋, 顾沛雯.2008.巢式PCR (Nested-PCR)在植原体检测中的应用[J].陕西农业科学, (03): 50-52.
(An F Q,Wu Y F,Gu P W.2008.Application of Nested-PCR in phytoplasma detection[J].Shaanxi Journal of Agricultural Sciences, (03): 50-52.)
[2] 范国权, 韩树鑫, 白艳菊, 等.2015.植原体病害研究进展[J].黑龙江农业科学, (11): 148-153.
(Fan G Q, Han S X, Bai Y J, et al.2015.Study advances of phytoplasma[J].Heilongjiang Agricultural Sciences, (11): 148-153.)
[3] 高洪.2010.剑麻的性能及应用[J].山东纺织科技, 51(03): 44-45.
(Gao H.2010.Property and application of sisal[J].Shandong Textile Science & Technology, 51(03): 44-45.)
[4] 胡盛红, 郑金龙, 温衍生, 等.2014.2013年中国剑麻产业形势分析及发展趋势[J].热带农业科学, 34(12): 111-117.
(Hu S H, Zheng J L, Wen Y S, et al.2014.Sisal industry situation and development trend in China in 2013[J].Chinese Journal of Tropical Agriculture, 34(12): 111-117.)
[5] 黄标, 夏李虹, 李江平, 等.2017.几种营养液药液及脱毒粉蚧对剑麻紫色卷叶病的影响[J].热带农业科学, 37(07): 41-45, 50.
(Huang B, Xia L H, Li J P, et al.2017.The Effect of liquid nutrient, liquid pesitcides and virus-free mealybugs on sisal purple leafroll disease[J].Chinese Journal of Tropical Agriculture, 37(07): 41-45, 50.)
[6] 梁宏合, 杜国冬, 王春田.2012.我国剑麻主要病虫害研究进展[J].广东农业科学, 39(22): 84-87.
(Liang H H, Du G D, Wang C T.2012.Research progress on main diseases and insect pests of sisal in China[J].Guangdong Agricultural Sciences, 39(22): 84-87.)
[7] 马占鸿, 黄冲, 孙振宇, 等.2011.小麦条锈菌单管一步巢式PCR检测方法及引物[P].中国, CN201110043132.9.
(Ma Z H, Huang C, Sun Z Y, et al.2011.Wheat stripe rust step single-tube nested PCR detection methods and primers[P].China, CN201110043132.9.)
[8] 闫伟, 李葱葱, 夏蔚, 等.2016.应用单管巢式和半巢式PCR检测转基因玉米MON89034[J].玉米科学, 24(04): 56-60.
(Yan W, Li C C, Xia W, et al.2016.Event-specific detection of genetically modified maize MON89034 based on the single tube nested and semi-nested PCR[J].Journal of Maize Sciences, 24(04): 56-60.)
[9] 杨坤, 高志鹏, 习丙文, 等.2020.洪湖碘泡虫单管半巢式PCR检测方法的建立及应用[J].中国水产科学, 27(08): 927-933.
(Yang K, Gao Z P, Xi B W, et al.2020.Development a single-tube, semi-nested PCR method for the detection of Myxobolus honghuensis (Myxoporea: Bivalvulida)[J].Journal of Fishery Sciences of China, 27(08): 927-933.)
[10] 赵艳龙, 何衍彪, 詹儒林.2007.我国剑麻主要病虫害的发生与防治[J].中国麻业科学, (06): 334-338.
(Zhao Y L, He Y B, Zhan R L.2007.The occurrence and control of the main diseases and pests in sisal hemp in China[J].Plant Fiber Sciences in China, (06): 334-338.)
[11] Contaldo N, Bertaccini A, Paltonieri S, et al.2012.Axenic culture of plant pathogenic phytoplasmas[J].Phytopathologia Mediterranea, 51(3): 607-617.
[12] Contaldo N, Satta E, Zambon Y, et al.2016.Development and evaluation of different complex media for phytoplasma isolation and growth[J].Journal of microbiological methods, 127: 105-110.
[13] Dey K K, Lin H, Borth W B, et al.2012.A highly sensitive single-tube nested PCR assay for the detection of pineapple mealybug wilt associated virus-2 (PMWaV-2)[J].Journal of Virological Methods, 183(2): 215-218.
[14] Diala S, Asmaa T, Yaqoub A.2020.Improving sensitivity of single tube nested PCR to detect fastidious microorganisms.[J].Heliyon, 6(1): e03246.
[15] Domingues L F, Botura M B, Cruz A C, et al.2010.Evaluation of anthelmintic activity of liquid waste of Agave sisalana (sisal) in goats[J].Revista Brasilra de Parasitologia Veterinária, 19(4): 270-272.
[16] Dunder R J, Quaglio A E, Maciel R P, et al.2010.Anti-inflammatory and analgesic potential of hydrolyzed extract of Agave sisalana Perrine ex Engelm, Asparagaceae[J].Revista Brasileira de Farmacognosia-brazilian Journal of Pharmacognosy, 20(3): 376-381.
[17] Firrao G, Andersen M, Bertaccini A, et al.2004.'Candidatus Phytoplasma', a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects[J].International Journal of Systematic & Evolutionary Microbiology, 54(4): 1243-1255.
[18] Gookin J L, Birkenheuer A J, Breitschwerdt E B, et al.2002.Single-tube nested PCR for detection of Tritrichomonas foetus in feline feces[J].Journal of Clinical Microbiology, 40(11): 4126-4130.
[19] Harrison N A, Davis R E, Oropeza C, et al.2014.'Candidatus Phytoplasma palmicola', associated with a lethal yellowing-type disease of coconut (Cocos nucifera L.) in Mozambique[J].International Journal of Systematic and Evolutionary Microbiology, 64(Pt 6): 1890-1899.
[20] Klemsdal S S, Elen O.2010.Development of a highly sensitive nested-PCR method using a single closed tube for detection of Fusarium culmorum in cereal samples[J].Letters in Applied Microbiology, 42(5): 544-548.
[21] Lee I M.1993.Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasmalike organisms[J].Phytopathology, 83(8): 834-842.
[22] Li Shu, Hao Weijia, Lu Guanghua, et al.2015.Occurrence and identification of a new vector of rice orange leaf Phytoplasma in South China[J].Plant Disease, 99(11): 1483-1487.
[23] Pérez-López E, Olivier C Y, Luna-Rodríguez M, et al.2016.Phytoplasma classification and phylogeny based on in silico and in vitro RFLP analysis of cpn60 universal target sequences[J].International Journal of Systematic and Evolutionary Microbiology, 66(12): 5600-5613.
[24] Romero J C L, Zavala J F A, Aguilar G A G, et al.2018.Biological activities of Agave by‐products and their possible applications in food and pharmaceuticals[J].Journal of the Science of Food and Agriculture, 98(7): 2461-2474.
[25] Santos J D G, Branco A, Silva A F, et al.2009.Antimicrobial activity of Agave sisalana[J].African Journal of Biotechnology, 8(22): 6181-6184.
[26] Sun Y, Chen J, Li J, et al.2017.Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of Tuberculous meningitis[J].PLOS ONE, 12(10): e0186985.
[27] Thongsri Y, Wonglakorn L, Chaiprasert A, et al.2013.Evaluation for the clinical diagnosis of pythium insidiosum using a single-tube nested PCR[J].Mycopathologia, 176(5-6): 369-376.
[28] Wei W, Davis R E, Lee I, et al.2007.Computer-simulated RFLP analysis of 16S rRNA genes: Identification of ten new phytoplasma groups[J].International Journal of Systematic and Evolutionary Microbiology, 57(8): 1855-1867.
[29] Yourno J.1997.Method and apparatus for nested polymerase chain reaction (PCR) with single closed reaction tubes[J].Biotechnology Advances, 15(2): 443. |
|
|
|
