基于TaqMan MGB探针的山茶叶杯菌实时荧光PCR快速检测方法

doi:10.16420/j.issn.0513-353x.2020-1027

摘要/Abstract

摘要：

对山茶叶杯菌（Ciborinia camelliae L. M. Kohn）及其近似种的β-微管蛋白（β-tubulin）基因序列进行了比较分析,设计了1对特异性引物和1条探针,建立了山茶叶杯菌实时荧光PCR检测方法。该方法能特异性检测山茶叶杯菌;体系最佳引物浓度、探针浓度分别为0.8和0.7 μmol · L^-1;在10 μL反应体系中,最低检测限可达1.0 pg;该方法可用于疑似携带山茶叶杯菌样品的检测与初筛。

关键词: 山茶, 花腐病, 山茶叶杯菌, TaqMan MGB探针, 实时荧光PCR, 快速检测

Abstract:

Based on β-tubulin gene of Ciborinia camelliae L. M. Kohn and related isolates,a pair of specific primers and a TaqMan MGB probe were designed and synthesized. A novel real-time fluorescent PCR was established to detect C. camelliae. The optimized primer and probe concentrations were 0.8 and 0.7 μmol · L^-1,respectively. The minimum detection limit of this method is up to 1.0 pg for 10 μL reaction system. The method was specific,sensitive,easily to operate and fast,which provide a valuable tool for early rapid detection and identification of C. camelliae.

Key words: Camellia, camellia flower blight, Ciborinia camelliae, TaqMan MGB probe, real-time fluorescent PCR, rapid detection

中图分类号:

S685.14

吕燕, 段维军. 基于TaqMan MGB探针的山茶叶杯菌实时荧光PCR快速检测方法[J]. 园艺学报, 2022, 49(3): 663-670.

LÜ Yan, DUAN Weijun. TaqMan MGB-based Real-time Fluorescence PCR Method for the Rapid Detection of Ciborinia camelliae[J]. Acta Horticulturae Sinica, 2022, 49(3): 663-670.

图/表 8

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来源地 Location	菌种名 Strain name	菌株编号 Strain number	寄主 Host	qRT-PCR检测结果 Result of qRT-PCR
日本Japan	山茶叶杯菌 Ciborinia camelliae	CM96 ~ CM104,CM115 ~ CM143,CM188,CM189	茶梅 Camellia sasanqua	阳性Positive
		CM105 ~ CM114	山茶Camellia japonica	阳性Positive
	叶点霉属Phyllosticta sp.	G1014,G1024	茶梅C. sasanqua	阴性Negative
		G1015	山茶C. japonica	阴性Negative
	刺盘孢属Colletotrichum sp.	CL1085,CL1086,CL1091 ~ CL1094	茶梅 C. sasanqua	阴性Negative
	间座壳属 Diaporthe sp.	P1102	茶梅 C. sasanqua	阴性Negative
	葡萄座腔菌属 Botryosphaeria sp.	BS1070,BS1072,BS1074	茶梅 C. sasanqua	阴性Negative
	茎点霉属Phoma sp.	PH1113,PH1119,PH1120	茶梅C. sasanqua	阴性Negative
	灰葡萄孢 Botrytis cinerea	BC	茶梅 C. sasanqua	阴性Negative
	杜鹃花枯萎病菌 Ovulinia azaleae	OV60	杜鹃 Rhododendron simsii Planch	阴性Negative
青海西宁 Xining,Qinghai	美澳型核果褐腐病菌 Monilinia fructicola	MF	梨Pear	阴性Negative
巴基斯坦Pakistan	桃褐腐核盘菌Monilinia laxa	ML	樱桃 Cerasus seudocerasus	阴性Negative
美国 American	核盘菌 Sclerotinia sclerotiorum	SS1	大豆 Glycine max	阴性Negative
加拿大Canada	麦角菌 Claviceps purpurea	SC1,SC3	大麦 Hordeum vulgare	阴性Negative
法国 France	麦角菌 C. purpurea	SC2	大麦 H. vulgare	阴性Negative

来源地 Location	菌种名 Strain name	菌株编号 Strain number	寄主 Host	qRT-PCR检测结果 Result of qRT-PCR
日本Japan	山茶叶杯菌 Ciborinia camelliae	CM96 ~ CM104,CM115 ~ CM143,CM188,CM189	茶梅 Camellia sasanqua	阳性Positive
		CM105 ~ CM114	山茶Camellia japonica	阳性Positive
	叶点霉属Phyllosticta sp.	G1014,G1024	茶梅C. sasanqua	阴性Negative
		G1015	山茶C. japonica	阴性Negative
	刺盘孢属Colletotrichum sp.	CL1085,CL1086,CL1091 ~ CL1094	茶梅 C. sasanqua	阴性Negative
	间座壳属 Diaporthe sp.	P1102	茶梅 C. sasanqua	阴性Negative
	葡萄座腔菌属 Botryosphaeria sp.	BS1070,BS1072,BS1074	茶梅 C. sasanqua	阴性Negative
	茎点霉属Phoma sp.	PH1113,PH1119,PH1120	茶梅C. sasanqua	阴性Negative
	灰葡萄孢 Botrytis cinerea	BC	茶梅 C. sasanqua	阴性Negative
	杜鹃花枯萎病菌 Ovulinia azaleae	OV60	杜鹃 Rhododendron simsii Planch	阴性Negative
青海西宁 Xining,Qinghai	美澳型核果褐腐病菌 Monilinia fructicola	MF	梨Pear	阴性Negative
巴基斯坦Pakistan	桃褐腐核盘菌Monilinia laxa	ML	樱桃 Cerasus seudocerasus	阴性Negative
美国 American	核盘菌 Sclerotinia sclerotiorum	SS1	大豆 Glycine max	阴性Negative
加拿大Canada	麦角菌 Claviceps purpurea	SC1,SC3	大麦 Hordeum vulgare	阴性Negative
法国 France	麦角菌 C. purpurea	SC2	大麦 H. vulgare	阴性Negative

DNA/（ng · μL^-1）	Ct
DNA/（ng · μL^-1）	重复1 Repeat 1	重复2 Repeat 2	重复3 Repeat 3	平均值 Mean	标准偏差 SD	变异系数/% CV
25.000	21.49	21.47	21.80	21.59	0.19	0.86
2.500	24.43	24.45	24.34	24.41	0.06	0.24
0.250	27.62	28.09	27.73	27.81	0.25	0.88
0.025	31.60	31.34	31.56	31.50	0.14	0.44

DNA/（ng · μL^-1）	Ct
DNA/（ng · μL^-1）	重复1 Repeat 1	重复2 Repeat 2	重复3 Repeat 3	平均值 Mean	标准偏差 SD	变异系数/% CV
25.000	21.49	21.47	21.80	21.59	0.19	0.86
2.500	24.43	24.45	24.34	24.41	0.06	0.24
0.250	27.62	28.09	27.73	27.81	0.25	0.88
0.025	31.60	31.34	31.56	31.50	0.14	0.44