The Rapid Detection of Xanthomonas citri ssp. citri（Xcc）Based on Recombinase Polymerase Amplification（RPA）Assay

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

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (3): 590-599.doi: 10.16420/j.issn.0513-353x.2020-0545

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The Rapid Detection of Xanthomonas citri ssp. citri（Xcc）Based on Recombinase Polymerase Amplification（RPA）Assay

MA Zhimin, DUAN Yu, XU Jianjian, BIN Yu, ZHOU Changyong(), SONG Zhen()

Citrus Research Institute,Southwest University/Chinese Academy of Agricultural Sciences,Chongqing 400712,China

Received:2020-12-16 Online:2021-03-25 Published:2021-04-02
Contact: ZHOU Changyong,SONG Zhen E-mail:zhoucy@cric.cn;songzhen@cric.cn

Abstract

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

CLC Number:

S666

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.

Figures/Tables 8

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

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.

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.

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

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.

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.

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.

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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The Rapid Detection of Xanthomonas citri ssp. citri（Xcc）Based on Recombinase Polymerase Amplification（RPA）Assay

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