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城市绿地中立枯丝核菌和齐整小核菌的qPCR快速检测方法
doi: 10.11833/j.issn.2095-0756.20210801
Quantitative real-time PCR for rapid detection of Rhizoctonia solani and Sclerotium rolfsii in urban green space
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摘要:
目的 土传病原真菌立枯丝核菌Rhizoctonia solani和齐整小核菌Sclerotium rolfsii严重威胁园林绿化植物正常生长。建立针对这2种土壤病原真菌的快速定量检测方法。 方法 通过筛选2种病原菌特异性引物，优化反应条件。 结果 初步建立了2种病原菌的实时荧光定量PCR(qPCR)检测方法。引物ST-RS1/ITS4和SRITSF/SRITSR可以分别用于立枯丝核菌和齐整小核菌的qPCR检测，其灵敏度分别达24×106和22×106拷贝·L−1，2次重复反应的变异系数分别为3.37%~4.61%和0.66%~8.61%。对上海绿地土壤样品的检测结果表明：立枯丝核菌和齐整小核菌的检出率分别为100%和19%。 结论 建立的qPCR检测方法具有较强特异性、较高灵敏度和较强重复性，可以用于上海城市绿地土壤中立枯丝核菌和齐整小核菌的快速、有效定量检测。图2表5参29 Abstract:Objective This study aims to establish a rapid quantitative detection method for Rhizoctonia solani and Sclerotium rolfsii, 2 soil-borne pathogenic fungi that seriously threaten the normal growth of landscape plants in Shanghai. Method The reaction conditions were optimized by screening 2 pathogen specific primers. Result A quantitative real-time PCR(qPCR) method was established for the detection of the two pathogens. The primers ST-RS1/ITS4 and SRITSF/SRITSR could be used for qPCR detection of R. solani and S. rolfsii, with sensitivity of 24×106 and 22×106 copies·L−1, respectively. The coefficients of variation of the 2 repeated reactions were 3.37%−4.61% and 0.66%−8.61%, respectively. The detection results of soil samples in Shanghai green space showed that the detection rates of R. solani and S. rolfsii were 100% and 19%, respectively. Conclusion The established qPCR method has high specificity, sensitivity and repeatability, and can be used for rapid and effective quantitative detection of R. solani and S. rolfsii in Shanghai urban green soil. [Ch, 2 fig. 5 tab. 29 ref.] -
Key words:
- urban green space soil /
- Rhizoctonia solani /
- Sclerotium rolfsii /
- qPCR
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表 1 绿地土壤样品信息
Table 1. Information of green space soil samples
样品编号 绿地类型 采样位置 行政区 绿地名称 纬度(N) 经度(E) 1 道路绿地 金山区 朱平公路中央绿化带内 30°49′ 121°08′ 2 公园绿地 闵行区 闵行文化公园 31°10′ 121°21′ 3 公园绿地 金山区 亭林公园 30°20′ 121°18′ 4 道路绿地 金山区 亭卫公路 30°20′ 121°20′ 5 公园绿地 奉贤区 奉浦四季生态园 30°56′ 121°27′ 6 公园绿地 崇明区 崇明新城公园 31°37′ 121°25′ 7 公园绿地 黄浦区 复兴公园 31°13′ 121°27′ 8 道路绿地 徐汇区 虹梅路 31°08′ 121°24′ 9 道路绿地 普陀区 真北路 31°16′ 121°23′ 10 道路绿地 浦东新区 杨高中路 31°13′ 121°32′ 11 道路绿地 浦东新区 杨高北路 31°15′ 121°35′ 12 道路绿地 浦东新区 济阳路上浦路 31°08′ 121°29′ 13 道路绿地 浦东新区 杨高南路 31°11′ 121°31′ 14 道路绿地 青浦区 外青松公路 31°12′ 121°07′ 15 道路绿地 浦东新区 唐镇路 31°12′ 121°38′ 16 公园绿地 宝山区 炮台湾湿地公园 31°23′ 121°30′ 17 公园绿地 宝山区 淞南公园 31°20′ 121°28′ 18 道路绿地 闵行区 华宁路剑川路 31°01′ 121°23′ 19 道路绿地 杨浦区 殷行路 31°19′ 121°31′ 20 公园绿地 浦东新区 名人苑公园北门东侧 31°14′ 121°33′ 21 公园绿地 嘉定区 汇龙潭公园 31°22′ 121°14′ 
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表 2 用于立枯丝核菌和齐整小核菌qPCR的引物信息
Table 2. qPCR primer sets of R. solani and S. rolfsii
病原菌 引物名称 引物序列 (5ʹ→3ʹ) 产物大小/bp 退火温度/℃ 参考文献 立枯丝核菌 ST-RS1 AGTGTTATGCTTGGTTCCACT 187 60 [19] ITS4 TCCTCCGCTTATTGATATGC ITS1 TCCGTAGGTGAACCTGCGG 564 65 [20] GMRS-3 AGTGGAACCAAGCATAACACT 齐整小核菌 SCR-F CGTAGGTGAACCTGCGGA 540 54 [21] SCR-R CATACAAGCTAGAATCCC SRITSF TACACCTGTGAACCAACTG 465 52 [22] SRITSR CATACAAGCTAGAATCCC
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表 3 qPCR引物在线比对结果
Table 3. On-line blast results of qPCR primers
引物名称 目的序列
长度/bp匹配目的
序列数/条匹配非目的
序列数/条错配
率/%ST-RS1/ITS4 187 908 76 7.7 ITS1/GMRS-3 564 936 63 6.3 SRITSF/SRITSR 465 429 38 8.1 SCR-F/SCR-R 540 30 4 13.3
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表 4 质粒标准品和土壤样品测序比对结果
Table 4. Sequencing alignment result of plasmid standard and soil sample
引物 样品 同源菌株 基因登录号 相似度/% ST-RS1/ITS4 质粒标准品 R. solani BTRFB1 MZ158299 99.4 土壤样品 R. solani AG-F JQ343829 99.4 SRITSF/SRITSR 质粒标准品 S. rolfsii NTNM MT126473 99.5 土壤样品 S. rolfsii SR1 MG847186 98.8
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表 5 qPCR的重复性
Table 5. Repeatability of qPCR
病原菌 样品编号 第1次试验 第2次试验 变异系数/% Ct 拷贝数/(×106拷贝·L−1) Ct 拷贝数/(×106拷贝·L−1) 立枯丝核菌 4 31.74 3.09×101 31.60 3.25×101 3.57 5 30.54 6.45×101 30.56 6.15×101 3.37 6 29.49 1.23×102 29.51 1.17×102 3.54 10 30.98 4.91×101 31.03 4.60×101 4.61 12 29.71 1.07×102 29.74 1.01×102 4.08 齐整小核菌 3 33.76 2.16×102 33.53 2.44×102 8.61 6 35.15 1.02×102 35.09 1.05×102 2.05 14 35.04 1.08×102 35.05 1.07×102 0.66 16 36.55 4.77×101 36.63 4.57×101 3.03 18 35.72 7.46×101 35.67 7.66×101 1.87
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