基于重组酶聚合酶扩增技术（RPA）的柑橘溃疡病菌检测方法

doi:10.16420/j.issn.0513-353x.2020-0545

摘要/Abstract

摘要：

根据柑橘溃疡病菌（Xanthomonas citri ssp. citri,Xcc）基因组的保守序列设计特异引物,通过对引物浓度、反应温度和反应时间条件优化,建立了柑橘溃疡病菌重组酶聚合酶扩增（Recombinase polymerase amplification,RPA）检测方法。该方法无需PCR仪等复杂设备,在39 ℃恒温反应30 min即可完成检测过程,快速简便。该检测方法与其他柑橘病原无交叉反应,特异性强;检测灵敏度是普通PCR的100倍,与实时荧光定量PCR基本一致。对71个柑橘样品进行检测,RPA检测出溃疡病阳性样品22个,与PCR、实时荧光定量PCR检测结果一致。

关键词: 柑橘溃疡病, 重组酶聚合酶扩增, RPA, 检测

Abstract:

A detection assay based on recombinase polymerase amplification（RPA）for Xanthomonas citri ssp.citri（Xcc）was established by designing specific primers and optimizing concentration of primers,reaction temperature and reaction time. The method requires no complex equipment such as PCR apparatus,and can complete the detection process at 39 ℃ in 30 min,which is quick and simple. The detection method has strong specificity to Xcc with no cross reaction with other citrus pathogens. The RPA detection sensitivity was 100 times higher than that of ordinary PCR,which was the same as that of real-time quantitative PCR. In 71 citrus samples,22 samples were positive to canker detected by RPA,which was consistent with the results of PCR and real-time quantitative PCR.

Key words: citrus bacterial canker disease, recombinase polymerase amplification, detection

中图分类号:

S666

马志敏, 段玉, 许建建, 宾羽, 周常勇, 宋震. 基于重组酶聚合酶扩增技术（RPA）的柑橘溃疡病菌检测方法[J]. 园艺学报, 2021, 48(3): 590-599.

MA Zhimin, DUAN Yu, XU Jianjian, BIN Yu, ZHOU Changyong, SONG Zhen. The Rapid Detection of Xanthomonas citri ssp. citri（Xcc）Based on Recombinase Polymerase Amplification（RPA）Assay[J]. Acta Horticulturae Sinica, 2021, 48(3): 590-599.

图/表 8

表1 试验所用引物

Table 1 Primers used in the experiments

体系 System	引物 Primer	引物序列（5′-3′） Primer sequence	产物长度/bp Product length	参考文献 Reference
RPA	CA1	F:CAACAACACGTAAAAGATCACCCCGACCCC; R:GATGTGCTGGTCACCGTCGATCACGGCATCGC	209
RPA	CA2	F:GTTGGTGTCGTCGCTTGTATGGACTATAGT; R:GCCGCGCACGGGTGCAAAAAATCTTCAACTTC	232
PCR	canker	F:GAGTCGCCTACCGAGAAATCC; R:ACCACGGCAGGGTGAAGAC	220	唐科志等,2005
qPCR	Xac 07	F:TTGGTGTCGTCGCTTGTAT; R:CACGGGTGCAAAAAATCT	82	赵云等,2006

图1 柑橘不同病原侵染样品的PCR或RT-PCR检测 M:标准分子量;1:柑橘溃疡病菌;2:柑橘黄龙病菌;3:柑橘黄化脉明病毒;4:柑橘叶斑驳病毒;5:柑橘碎叶病毒;6:柑橘裂皮类病毒;7:柑橘鳞皮病毒;8:温州蜜柑萎缩病毒;9:柑橘衰退病毒。

Fig. 1 PCR or RT-PCR detection for samples infected by different citrus pathogens M:Standard molecular weight;1:Xanthomonas citrissp.citri;2:Candidatus Liberibacter asiaticus;3:Citrus yellow vein clearing virus;4:Citrus leaf blotch virus;5:Citrus tatter leaf virus;6:Citrus exocortis viroid;7:Citrus psorosis virus;8:Satsuma dwarf virus;9:Citrus tristeza virus.

图2 柑橘溃疡病菌RPA引物筛选 M:标准分子量;1、4:柑橘溃疡病菌阳性样品;2、5:柑橘溃疡病菌阴性样品;3、6:去离子水。

Fig. 2 Screening of Xanthomonas citri ssp. citri RPA primers M:Standard molecular weight;1,4:Xanthomonas citrissp.citri positive sample;2,5:Xanthomonas citrissp.citri negative sample;3,6:ddH2O.

图3 RPA扩增产物的序列比对 1:参考序列;2:测序结果。

Fig. 3 Sequence alignment of RPA amplification products 1:Reference sequence;2:Sequencing result.

图4 柑橘溃疡病菌RPA检测体系的优化 A:引物浓度筛选;B:反应温度筛选;C:反应时间筛选;M:标准分子量。

Fig. 4 Establishment of RPA assay for Xanthomonas citri ssp. citri detection A:Screening of primers concentration;B:Screening of reaction temperature;C:Screening reaction time;M:Standard molecular weight.

图5 RPA 检测柑橘溃疡病菌的特异性 M:标准分子量;1:柑橘溃疡病菌;2:柑橘黄龙病菌;3:柑橘黄化脉明病毒;4:柑橘叶斑驳病毒;5:柑橘碎叶病毒;6:柑橘裂皮类病毒;7:柑橘鳞皮病毒;8:温州蜜柑萎缩病毒;9:柑橘衰退病毒;10:去离子水;11:柑橘溃疡病菌阳性对照。

Fig. 5 Specificity of RPA assays for Xanthomonas citri ssp. citri detection M:Standard molecular weight;1:Xanthomonas citrissp.citri;2:Candidatus Liberibacter asiaticus;3:Citrus yellow vein clearing virus;4:Citrus leaf blotch virus;5:Citrus tatter leaf virus;6:Citrus exocortis viroid;7:Citrus psorosis virus;8:Satsuma dwarf virus;9:Citrus tristeza virus;10:ddH2O;11:Xanthomonas citri ssp. citri positive control.

图6 RPA、PCR和实时荧光定量PCR检测柑橘溃疡病菌的灵敏度 A:RPA;B:PCR;C:实时荧光定量PCR,M:标准分子量。

Fig. 6 Sensitivity of RPA,PCR and real-time quantitative PCR for Xanthomonas citri ssp. citri detection A:RPA;B:PCR;C:Real-time quantitative PCR;M:Standard molecular weight.

表2 田间样品的柑橘溃疡病菌RPA、PCR和实时荧光定量PCR检测

Table 2 Xanthomonas citri ssp. citri detection of field samples using RPA,PCR and Real-time quantitative PCR

品种 Variety	总样品数 Number of samples	阳性样品数（阳性率/%）Positive number（Positive rate）
品种 Variety	总样品数 Number of samples	RPA	PCR	实时荧光定量PCR Real-time quantitative PCR
十月橘 Shiyueju	8	8（100）	8（100）	8（100）
脐血橙Washington Sanguine	2	2（100）	2（100）	2（100）
大雅柑 Dayagan	2	2（100）	2（100）	2（100）
琯溪蜜柚Guanxi Miyou	7	0（0）	0（0）	0（0）
尤力克柠檬 Eureka Lemon	4	0（0）	0（0）	0（0）
沃柑 Orah	16	1（6.25）	1（6.25）	1（6.25）
不知火 Shiranui Tangor	18	1（5.56）	1（5.56）	1（5.56）
塔罗科血橙 Tarocco	14	8（57.14）	8（57.14）	8（57.14）
总计 Total	71	22（30.99）	22（30.99）	22（30.99）

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