Establishment and Application of Real-time PCR for Sugarcane Striate Virus

doi:10.13560/j.cnki.biotech.bull.1985.2024-0129

Abstract

Abstract:

【Objective】 It is to establish a rapid, sensitive and specific SYBR Green I quantitative PCR method for the detection of sugarcane striate virus（SStrV）.【Method】 A specific amplification primer was designed from the conserved sequence of the SStrV genome sequence, the recombinant plasmid pMD19-T-SStrV-qN containing SStrV gene was constructed as a positive plasmid standard. Using it as template, the SStrV fluorescence quantitative PCR assay was established. And the sensitivity, specificity, stability, and subsequently of this method were tested, then the SStrV loads in different tissue sites of sugarcane were detected.【Result】 The recombinant plasmid containing SStrV genome sequence was diluted into a standard at a 10-fold ratio, and they were used as a template for real-time PCR, the standard curve y = -3.337 x + 38.197 was obtained, and the correlation coefficient r² = 0.999 was obtained, indicating that the Cq value was linearly related to the logarithm of the copy number of the standard concentration. With this established real-time PCR, the least detection limit was 13 copies of the recombinant plasmid/μL, which was 100 times more sensitive than ordinary PCR. The method specifically detected SStrV with high specificity, and the coefficient of variation within and between groups was n 0.13%-0.94%, indicating that the method was of good repeatability. The load of SStrV was significantly different among different tissue sites of the sugarcane, and the load of SStrV was the highest in +4 leaves, which was significantly different from other tissue sites.【Conclusion】 The SYBR Green I quantitative PCR method is established to provide an efficient quantitative detection method for the diagnosis of SStrV, and it is determined that +4 leaves are the best sampling site for the detection of SStrV in sugarcane.

Key words: sugarcane striate virus, real-time PCR, viral load, detection

WANG Chao-min, HE Mei-dan, WANG Wen-zhi, YUAN Qian-hua, ZHANG Shu-zhen, SHEN Lin-bo. Establishment and Application of Real-time PCR for Sugarcane Striate Virus[J]. Biotechnology Bulletin, 2024, 40(6): 126-133.

Figures/Tables 6

Fig. 1 PCR amplification of SStrV fragments M：Marker DS2000; 1-2: amplified SStrV target fragment; 3: negative control

Fig. 2 PCR standard curve（A）, amplification curve（B）and melting curve（C） A is standard curve, built with a 10-fold serial dilution of SStrV plasmid DNA of 1.31 ×108 copies /μL constructed, respectively. B is amplification curve, from left to right were developed with a 10-fold serial dilution of SStrV plasmid DNA. C is melting curve, and its peak corresponds to a temperature greater than 80℃

Fig. 3 Amplification plot of the specificity assay 1：SStrV. 2-6：SCBV、SCMV、SCSMV、SrMV、SCYLV. 7：Blank control

Fig. 4 Amplification curve for qPCR sensitivity experiments（A） and sensitivity analysis of conventional PCR（B） A: From left to right were developed with a 10-fold serial dilution of SStrv plasmid DNA; B: M: DSTM 2000（marker）；1-8: 1.31×108 copies /μL-1.31×10 copies /μL. 9: Blank control

Table 1 Results of qPCR repeatability test

质粒浓度Plasmid concentration/（copies·µL^-1）	组内实验Within-group experiments		组间实验Intergroup experiments
质粒浓度Plasmid concentration/（copies·µL^-1）	Ct值（x±s） Ct value	变异系数（CV） Coefficient variation/%	Ct值（x±s） Ct value	变异系数（CV） Coefficient variation/%
1.31×10⁸	10.94±0.09	0.81	10.89±0.10	0.94
1.31×10⁷	14.51±0.02	0.13	14.44±0.13	0.92
1.31×10⁶	17.90±0.06	0.31	17.80±0.15	0.87
1.31×10⁵	21.35±0.08	0.37	21.23±0.16	0.74
1.31×10⁴	24.47±0.11	0.43	24.53±0.11	0.45
1.31×10³	27.49±0.13	0.48	27.51±0.17	0.63

Fig. 5 Detection of SStrV in different tissue parts of eight lines The error line in the figure refers to the standard deviation. The lowercase letters indicates that the SStrV load of the virus in different tissue sites is significantly different（P <0.05）（Calculated using the Duncan method）

References 35

[1]	Kandel R, Yang XP, Song J, et al. Potentials, challenges, and genetic and genomic resources for sugarcane biomass improvement[J]. Front Plant Sci, 2018, 9: 151. doi: 10.3389/fpls.2018.00151 pmid: 29503654
[2]	Apon TA, Ahmde SF, Bony ZF, et al. Sett priming with salicylic acid improves salinity tolerance of sugarcane(Saccharum officinarum L.) during early stages of crop development[J]. Heliyon, 2023, 9(5): e16030.
[3]	张跃彬, 吴才文. 国内外甘蔗产业技术进展及发展分析[J]. 中国糖料, 2017, 39(3): 47-50.
	Zhang YB, Wu CW. Progress and development analysis of sugarcane industry in the world[J]. Sugar Crops China, 2017, 39(3): 47-50.
[4]	Swapna M, Kumar S. MicroRNAs and their regulatory role in sugarcane[J]. Front Plant Sci, 2017, 8: 997. doi: 10.3389/fpls.2017.00997 pmid: 28659947
[5]	黄佳艳, 冯小艳, 沈林波, 等. 甘蔗ShPR10基因的克隆及其编码蛋白与甘蔗线条花叶病毒P1蛋白的互作研究[J]. 生物技术通报, 2023, 39(10): 163-174. doi: 10.13560/j.cnki.biotech.bull.1985.2023-0403
	Huang JY, Feng XY, Shen LB, et al. Cloning of sugarcane ShPR10 gene and study on the interaction between ShPR10 protein and P1 protein encoded by sugarcane streak mosaic virus[J]. Biotechnol Bull, 2023, 39(10): 163-174.
[6]	Calderan-Rodrigues MJ, de Barros Dantas LL, Cheavegatti Gianotto A, et al. Applying molecular phenotyping tools to explore sugarcane carbon potential[J]. Front Plant Sci, 2021, 12: 637166.
[7]	李明, 田洪春, 黄智刚. 我国甘蔗产业发展现状研究[J]. 中国糖料, 2017, 39(1): 67-70.
	Li M, Tian HC, Huang ZG. Research on the development status of sugarcane industry in China[J]. Sugar Crops China, 2017, 39(1): 67-70.
[8]	价格成本调查中心. 2022/23 年制糖期行业运行情况及新制糖期展望[EB/OL].[2023-12-26]. https://www.ndrc.gov.cn/wsdwhfz/202312/t20231226_1362936.html.
	Price Cost Survey Center. Industry operation in 2022/23 sugar production period and outlook for new sugar production period[EB/OL].[2023-12-26]. https://www.ndrc.gov.cn/wsdwhfz/202312/t202312261362936.html.
[9]	许孚, 汪洲涛, 路贵龙, 等. 甘蔗遗传改良中的基因工程:适用、成就、局限和展望[J]. 农业生物技术学报, 2022, 30(3): 580-593.
	Xu F, Wang ZT, Lu GL, et al. Genetic engineering in sugarcane improvement: adaptability, achievements, limitations and prospects[J]. J Agric Biotechnol, 2022, 30(3): 580-593.
[10]	Gallan DZ, Penteriche AB, Henrique MO, et al. Sugarcane multitrophic interactions: integrating belowground and aboveground organisms[J]. Genet Mol Biol, 2022, 46(Suppl 1): e20220163.
[11]	许小洁, 于伟鹏, 杨广玲, 等. 甘蔗花叶病毒辅助成分-蛋白酶基因原核表达及抗血清制备[J]. 山东农业科学, 2019, 51(3): 87-91.
	Xu XJ, Yu WP, Yang GL, et al. Prokaryotic expression and antiserum preparation of helper component-proteinase of sugarcane mosaic virus[J]. Shandong Agric Sci, 2019, 51(3): 87-91.
[12]	陈海, 申亚南, 吕文竹, 等. 甘蔗高粱花叶病毒RT-LAMP快速检测方法的建立及评价[J]. 分子植物育种, 2020, 18(10): 3282-3287.
	Chen H, Shen YN, Lü WZ, et al. Establishment and evaluation of RT-LAMP for the rapid detection of Sorghum mosaic virus[J]. Mol Plant Breed, 2020, 18(10): 3282-3287.
[13]	徐小伟. 甘蔗线条花叶病毒P1蛋白诱导的广谱抗病蛋白的鉴定及功能研究[D]. 扬州: 扬州大学, 2023.
	Xu XW. Identification and functional analysis of broad-spectrum resistance protein induced by P1 protein of sugarcane streak mosaic virus[D]. Yangzhou: Yangzhou University, 2023.
[14]	许跃语. 甘蔗响应黄叶病毒侵染的转录组差异表达研究[D]. 福州: 福建农林大学, 2019.
	Xu YY. Transcriptomic analysis of differentially expressed genes in sugarcane in response to yellow leaf virus infection[D]. Fuzhou: Fujian Agriculture and Forestry University, 2019.
[15]	孙生仁, 张慧丽, 吴小斌, 等. 甘蔗杆状病毒及其启动子功能的研究进展[J]. 植物生理学报, 2018, 54(6): 943-950.
	Sun SR, Zhang HL, Wu XB, et al. Research advances in sugarcane bacilliform virus and its encoded promoter[J]. Plant Physiol J, 2018, 54(6): 943-950.
[16]	周雪平, 崔晓峰, 陶小荣. 双生病毒—— 一类值得重视的植物病毒[J]. 植物病理学报, 2003, 33(6): 487-492.
	Zhou XP, Cui XF, Tao XR. Geminiviruses—an emerging threat for crop production[J]. Acta Phytopathol Sin, 2003, 33(6): 487-492.
[17]	李静. 我国六种双生病毒的分子鉴定及两种病毒的致病性研究[D]. 杭州: 浙江大学, 2010.
	Li J. Molecular identification of six begomoviruses and pathogenicity of two begomoviruses in china[D]. Hangzhou: Zhejiang University, 2010.
[18]	Kraberger S, Saumtally S, Pande D, et al. Molecular diversity, geographic distribution and host range of monocot-infecting mastreviruses in Africa and surrounding islands[J]. Virus Res, 2017, 238: 171-178. doi: S0168-1702(17)30377-5 pmid: 28687345
[19]	Lin YF, Ali N, Hajimorad MR, et al. Incidence, geographical distribution, and genetic diversity of sugarcane striate virus in Saccharum species in China[J]. Plant Dis, 2021, 105(11): 3531-3537.
[20]	Filloux D, Fernandez E, Comstock JC, et al. Viral metagenomic-based screening of sugarcane from Florida reveals occurrence of six sugarcane-Infecting viruses and high prevalence of sugarcane yellow leaf virus[J]. Plant Dis, 2018, 102(11): 2317-2323. doi: 10.1094/PDIS-04-18-0581-RE pmid: 30207899
[21]	Boukari W, Alcalá-Briseño RI, Kraberger S, et al. Occurrence of a novel mastrevirus in sugarcane germplasm collections in Florida, Guadeloupe and Réunion[J]. Virol J, 2017, 14(1): 146.
[22]	于志亚, 杨鸣发, 马云云, 等. SFTSV实时荧光定量RT-PCR检测方法的建立及应用[J]. 中国兽医科学, 2022, 52(3): 292-297.
	Yu ZY, Yang MF, Ma YY, et al. Establishment and application of real-time fluorescent quantitative RT-PCR for the detection of SFTSV[J]. Chin Vet Sci, 2022, 52(3): 292-297.
[23]	张记宇, 韩郁茹, 时洪艳, 等. 猪急性腹泻综合征冠状病毒SYBR Green荧光定量PCR检测方法的建立及应用[J]. 畜牧兽医学报, 2021, 52(10): 2887-2894. doi: 10.11843/j.issn.0366-6964.2021.010.019
	Zhang JY, Han YR, Shi HY, et al. Establishment and application of SYBR green real-time PCR for swine acute diarrhea syndrome coronavirus[J]. Acta Vet Zootechnica Sin, 2021, 52(10): 2887-2894.
[24]	王坤, 陈开闯, 胡筱璇, 等. 甘蔗杆状病毒荧光定量PCR检测方法的建立及初步应用[J]. 基因组学与应用生物学, 2020, 39(4): 1779-1784.
	Wang K, Chen KC, Hu XX, et al. Establishment and preliminary application of fluorescence quantitative PCR for detection of sugarcane bacilliform virus[J]. Genom Appl Biol, 2020, 39(4): 1779-1784.
[25]	王洪星, 龚殿, 张雨良, 等. 甘蔗黄叶病毒实时荧光RT-PCR检测体系研究[J]. 中国糖料, 2013, 35(1): 1-4.
	Wang HX, Gong D, Zhang YL, et al. Research of real-time RT-PCR detection system for sugarcane yellow leaf virus[J]. Sugar Crops China, 2013, 35(1): 1-4.
[26]	王会敏. 甘蔗抗甘蔗条纹花叶病毒精准评价体系的建立与应用[D]. 南京: 南京农业大学, 2022.
	Wang HM. Establishment and application of an accurate evaluation system for sugarcane resistance to Sugarcane streak mosaic virus[D]. Nanjing: Nanjing Agricultural University, 2022.
[27]	王洪星. 侵染海南甘蔗的高粱花叶病毒鉴定及检测[D]. 海口: 海南大学, 2012.
	Wang HX. Characterization and detection of sorghum mosaic virus of sugarcane in Hainan[D]. Haikou: Hainan University, 2012.
[28]	淡明, 李松, 余坤兴, 等. 甘蔗健康种苗宿根矮化病的荧光定量PCR检测[J]. 中国农学通报, 2011, 27(5): 372-376.
	Dan M, Li S, Yu KX, et al. Detection of ratoon stunting disease in virus-free seedcane of sugarcane by real-time fluorescence quantitative PCR[J]. Chin Agric Sci Bull, 2011, 27(5): 372-376. doi: 10.11924/j.issn.1000-6850.2010-2614
[29]	Garces FF, Gutierrez A, Hoy JW. Detection and quantification of Xanthomonas albilineans by qPCR and potential characterization of sugarcane resistance to leaf scald[J]. Plant Dis, 2014, 98(1): 121-126. doi: 10.1094/PDIS-04-13-0431-RE pmid: 30708616
[30]	夏宝山. 我国蔗区甘蔗杆状病毒的发生及其qPCR和RCA检测方法的建立与应用[D]. 海口: 海南大学, 2023.
	Xia BS. The occurrence of Sugarcane bacilliform virus in sugarcane planting areas of China and the establishment and application of qPCR and RCA detection methods[D]. Haikou: Hainan University, 2023.
[31]	Wang D, Zhang X, Chen DX, et al. First report of Sida leaf curl virus and associated Betasatellite from tobacco[J]. Plant Dis, 2022, 106(3): 1078.
[32]	Wang D, Zhang X, Yao XM, et al. A 7-amino-acid motif of rep protein essential for virulence is critical for triggering host defense against Sri Lankan cassava mosaic virus[J]. Mol Plant Microbe Interact, 2020, 33(1): 78-86.
[33]	Wang D, Zhang X, Yin YT, et al. First report of Ludwigia yellow vein vietnam virus causing leaf curling on tobacco plants in Hainan province, China[J]. Plant Dis, 2023,107:2559.
[34]	Wang N, Zhao PZ, Wang D, et al. Diverse begomoviruses evolutionarily hijack plant terpenoid-based defense to promote whitefly performance[J]. Cells, 2022, 12(1): 149.
[35]	Ye J, Zhang LL, Zhang X, et al. Plant defense networks against insect-borne pathogens[J]. Trends Plant Sci, 2021, 26(3): 272-287. doi: 10.1016/j.tplants.2020.10.009 pmid: 33277186