番茄褪绿病毒(ToCV)石家庄分离物全基因组序列特征及荧光定量检测方法的建立

doi:10.7668/hbnxb.20196175

摘要/Abstract

摘要：

为准确鉴定番茄褪绿病毒病致病来源及河北省石家庄地区番茄褪绿病毒(ToCV)的分类地位,分析了采集于石家庄大河镇番茄种植基地病样进化发生关系,同时建立了ToCV高效鉴定方法。选取NCBI数据库中已公布的ToCV序列进行相似性分析,并将ToCV的RNA1和RNA2全长序列、热激蛋白70同系物(Hsp70h)和外壳蛋白(CP)序列构建系统进化树。结果显示,ToCV-SJZ分离物的RNA1和RNA2序列与ToCV-XS台湾株系亲缘关系最近,序列相似性分别为85.2%,92.4%。进化分析表明,ToCV株系可分为两大类,其中大多数ToCV株系与ToCV-Florida株系形成一个大分支,属于ToCV-Florida株系;而ToCV-SJZ株系单独与ToCV-XS台湾株系归类为一个小的分支,属于ToCV-XS台湾株系。根据进化分支代表性株系ToCV-SJZ、ToCV-XS和ToCV-Florida的CP基因序列,分别设计了可单独和复合检测2个株系的特异性RT-qPCR引物,建立了基于SYBR Green Ⅰ的实时荧光定量PCR检测体系。综上所述,明确了ToCV-SJZ分离物的分类地位,也为抗病种质资源鉴定及科学防控提供技术支持。

关键词: 番茄褪绿病毒(ToCV), 序列分析, 进化分析, SYBR Green Ⅰ荧光定量PCR

Abstract:

In order to identify the pathogenic source of Tomato chlorosis virus disease and clarify the taxonomic status of Tomato chlorosis virus (ToCV)in the Shijiazhuang area of Hebei Province,an analysis of the phylogenetic relationships of diseased samples collected from the Tomato Planting Base in Dahe Town,Shijiazhuang,was conducted.Additionally,an efficient method for ToCV identification was established.The published ToCV sequences in NCBI database were selected for similarity analysis,and the full-length RNA1 and RNA2 sequences of ToCV,Heat-shock protein 70 homologue(Hsp70h)and coat protein(CP)sequences were used to construct the phylogenetic tree.The results showed that the nucleotide(nt)sequences of RNA1 and RNA2 of the ToCV-SJZ shared 85.2% and 92.4% similarity,respectively,with the corresponding sequences of the ToCV-XS Taiwan strain,indicating the closest genetic relationship.The evolutionary analysis showed that ToCV could be divided into two clades,one represented by the Florida strain and the other by the Taiwan strain.According to the CP gene sequences of ToCV-SJZ,ToCV-XS and ToCV-Florida strains,a RT-qPCR assay system based on SYBR Green Ⅰ was established.In conclusion,this study clarifies the taxonomic status of the ToCV-SJZ isolate and provides technical support for the identification of disease-resistant germplasm resources and scientific prevention and control.

Key words: Tomato chlorosis virus(ToCV), Sequence analysis, Phylogenetic analysis, SYBR Green Ⅰ Real-time PCR

中图分类号:

康忱, 王鹏, 高亢, 田哲娟, 郝玲玉, 李亚栋, 吴志明. 番茄褪绿病毒(ToCV)石家庄分离物全基因组序列特征及荧光定量检测方法的建立[J]. 华北农学报, 2026, 41(3): 185-197. doi: 10.7668/hbnxb.20196175.

KANG Chen, WANG Peng, GAO Kang, TIAN Zhejuan, HAO Lingyu, LI Yadong, WU Zhiming. Complete Genomic Sequence Characteristics of Tomato chlorosis virus Shijiazhuang Isolate and Establishment of qPCR Detection Technique[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(3): 185-197. doi: 10.7668/hbnxb.20196175.

http://www.hbnxb.net/CN/Y2026/V41/I3/185

图/表 12

表1 本研究中所用的引物

Tab.1 Primers used in this study

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	基因组位置 Location	产物大小/bp Product size	用途 Function
ToCV-CP-491-F	ACACTCCACTAGGGGTGATACTCTT	RNA2-4548	491	ToCV检测
ToCV-CP-491-R	AAGCGTTTCTTTTCATAAGCAGGTT	RNA2-5038
SJZ-RNA1-Ⅰ-F	GAAATAGTATTCGTGTGATTACACA	RNA1-1	2 078	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅰ-R	GTCCTTAAGTCCCCACATGGTATCG	RNA1-2078
SJZ-RNA1-Ⅱ-F	CTATGCTCGATACCATGTGGGGACT	RNA1-2046	2 088	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅱ-R	GTTTCATCCAATTCTGGCAACTTTT	RNA1-4133
SJZ-RNA1-Ⅲ-F	TTGGAGCGAAGATTTCTTGGTA	RNA1-3979	1 474	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅲ-R	CCATATCCGTGTCCATTTTACC	RNA1-5452
SJZ-RNA1-Ⅳ-F	GGAAAAACGACCACGATAGTAAAGA	RNA1-5254	1 848	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅳ-R	CCACTGATACCACACCTACCACG	RNA1-7101
SJZ-RNA1-Ⅴ-F	CAAGGTATTCTTTTCAAGGTTTATG	RNA1-6974	1 623	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅴ-R	CGACCTATTTATTTATATACTAGATCTACC	RNA1-8596
SJZ-RNA2-Ⅰ-F	GAAATACTAGTCCAGGTGTTTCCTG	RNA2-1	1 765	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅰ-R	TTATGAGTGACAGGCTATGATTGC	RNA2-1765
SJZ-RNA2-Ⅱ-F	TAACATAATAAGTGATGACGGAAG	RNA2-1536	1 910	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅱ-R	ACTCTCTTTATGAAACTCAACAACTC	RNA2-3445
SJZ-RNA2-Ⅲ-F	TGTGAATAGTAAAGGTTTCGGAGAT	RNA2-3365	1 829	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅲ-R	AAGTCACAGTCTGATACAAGAAGCC	RNA2-5193
SJZ-RNA2-Ⅳ-F	TAAGACAAATGGATGATAATGAGTTC	RNA2-5104	1 740	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅳ-R	ACGAGTTGAGAAATCTTGAGTGTG	RNA2-6843
SJZ-RNA2-Ⅴ-F	TTGCTGTAGTGACAACTCATCTTTC	RNA2-6746	1 497	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅴ-R	CGACCTATTTATTTATATACTAAATCTACCA	RNA2-8242
SJZ-CP-216-F	AGAGACGACGCAACTTCGCTTG	RNA2-4441	216	ToCV-SJZ CP基因特异性RT-qPCR
SJZ-CP-216-R	CTTATCAGTCAATGGAGTCTCACCG	RNA2-4656
Flo-CP-250-F	GAGGTAGATGGTGCGAGATTTAAG	RNA2-4753	250	ToCV-Florida CP基因特异性RT-qPCR
Flo-CP-250-R	GATCATCTGAGATATTCATCAACGA	RNA2-5002
ToCV-CP-154-F	GCACTCTACAAGGGGTGATAC	RNA2-4548	154	ToCV CP基因通用RT-qPCR
ToCV-CP-154-R	ATTTACAATCGCCTGACACAT	RNA2-4701

表1 本研究中所用的引物

Tab.1 Primers used in this study

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	基因组位置 Location	产物大小/bp Product size	用途 Function
ToCV-CP-491-F	ACACTCCACTAGGGGTGATACTCTT	RNA2-4548	491	ToCV检测
ToCV-CP-491-R	AAGCGTTTCTTTTCATAAGCAGGTT	RNA2-5038
SJZ-RNA1-Ⅰ-F	GAAATAGTATTCGTGTGATTACACA	RNA1-1	2 078	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅰ-R	GTCCTTAAGTCCCCACATGGTATCG	RNA1-2078
SJZ-RNA1-Ⅱ-F	CTATGCTCGATACCATGTGGGGACT	RNA1-2046	2 088	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅱ-R	GTTTCATCCAATTCTGGCAACTTTT	RNA1-4133
SJZ-RNA1-Ⅲ-F	TTGGAGCGAAGATTTCTTGGTA	RNA1-3979	1 474	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅲ-R	CCATATCCGTGTCCATTTTACC	RNA1-5452
SJZ-RNA1-Ⅳ-F	GGAAAAACGACCACGATAGTAAAGA	RNA1-5254	1 848	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅳ-R	CCACTGATACCACACCTACCACG	RNA1-7101
SJZ-RNA1-Ⅴ-F	CAAGGTATTCTTTTCAAGGTTTATG	RNA1-6974	1 623	ToCV-SJZ RNA1克隆
SJZ-RNA1-Ⅴ-R	CGACCTATTTATTTATATACTAGATCTACC	RNA1-8596
SJZ-RNA2-Ⅰ-F	GAAATACTAGTCCAGGTGTTTCCTG	RNA2-1	1 765	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅰ-R	TTATGAGTGACAGGCTATGATTGC	RNA2-1765
SJZ-RNA2-Ⅱ-F	TAACATAATAAGTGATGACGGAAG	RNA2-1536	1 910	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅱ-R	ACTCTCTTTATGAAACTCAACAACTC	RNA2-3445
SJZ-RNA2-Ⅲ-F	TGTGAATAGTAAAGGTTTCGGAGAT	RNA2-3365	1 829	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅲ-R	AAGTCACAGTCTGATACAAGAAGCC	RNA2-5193
SJZ-RNA2-Ⅳ-F	TAAGACAAATGGATGATAATGAGTTC	RNA2-5104	1 740	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅳ-R	ACGAGTTGAGAAATCTTGAGTGTG	RNA2-6843
SJZ-RNA2-Ⅴ-F	TTGCTGTAGTGACAACTCATCTTTC	RNA2-6746	1 497	ToCV-SJZ RNA2克隆
SJZ-RNA2-Ⅴ-R	CGACCTATTTATTTATATACTAAATCTACCA	RNA2-8242
SJZ-CP-216-F	AGAGACGACGCAACTTCGCTTG	RNA2-4441	216	ToCV-SJZ CP基因特异性RT-qPCR
SJZ-CP-216-R	CTTATCAGTCAATGGAGTCTCACCG	RNA2-4656
Flo-CP-250-F	GAGGTAGATGGTGCGAGATTTAAG	RNA2-4753	250	ToCV-Florida CP基因特异性RT-qPCR
Flo-CP-250-R	GATCATCTGAGATATTCATCAACGA	RNA2-5002
ToCV-CP-154-F	GCACTCTACAAGGGGTGATAC	RNA2-4548	154	ToCV CP基因通用RT-qPCR
ToCV-CP-154-R	ATTTACAATCGCCTGACACAT	RNA2-4701

表2 不同毛形病毒属病毒在GenBank中的收录情况

Tab.2 Accession codes of different criniviruses in GenBank

分离物 Isolate	年份 Year	来源 Source	RNA1 登录号 GenBank accession No.of RNA1	RNA2登录号 GenBank accession No.of RNA2
ToCV-PAK1	2019	巴基斯坦	MN869004.1	MN869006.1
ToCV-BR	2019	巴西	MT279194.1	MT279195.1
ToCV-PAP-JJ6	2023	韩国	OR865222.1	OR865223.1
ToCV-Florida	1998	美国	AY903447.1	AY903448.1
ToCR-186	2015	南非	KY471129.1	KY471130.1
ToCV-KGS	2020	日本	LC711108.1	LC711109.1
ToCV-Kas	2015	土耳其	KY419526.1	KY419528.1
ToCV-AT80/99-IC	2014	西班牙	KJ740256.1	KJ740257.1
ToCV-DSMZ PV-1242	2022	希腊	ON398512.1	ON398513.1
ToCV-FERA_160205	2016	英国	KY810786.1	KY810787.1
ToCV-BJ	2013	中国北京	KC887998.1	KC887999.1
ToCV-SDSG	2012	中国山东	KC709509.1	KC709510.1
ToCV-SDTA	2019	中国山东	OR246919.1	OR246920.1
ToCV-SH-SIA	2019	中国上海	MW490611.1	MW490612.1
ToCV-XS	2011	中国台湾	KY618796.1	KY618797.1
ToCV-CQ630	2019	中国重庆	PQ213439.1	PQ213440.1
BYVaV	2005	美国	NC_006962.1	NC_006963.1
CCYV	2009	中国台湾	KY618798.1	KY618799.1
CYSDV	2009	美国	EF547827.1	FJ492808.1
DVCV	2009	美国	GQ225585.1	GQ376201.1
LCV	2008	美国	NC_012909.1	NC_012910.1
LIYV	1994	美国	U15440.1	U15441.1
SPaV	2009	美国	AY488137.1	AY488138.1
SPCSV	2002	乌干达	AJ428554.1	AJ428555.1
TVCV	2011	韩国	KR002686.1	KR002687.1
TICV	2009	美国	FJ815440.1	FJ815441.1

图1 疑似感染ToCV番茄样品叶片症状及PCR扩增结果 A.疑似感染ToCV番茄叶片症状;B.疑似感染ToCV番茄PCR检测结果:M.DNA Marker,N.阴性对照,0.健康番茄样品,24-1~24-15.2024年番茄样品。

Fig.1 The typical symptoms of tomato infection with ToCV on leaves and PCR amplification of suspected infected tomato samples A.The typical symptoms of ToCV on leaves;B.PCR amplification of suspected infected tomato samples: M.DNA Marker,N.Negative control,0.Uninfected tomato sample,24-1-24-15.Tomato samples in 2024.

图2 ToCV-SJZ 分离物RNA1和RNA2全长扩增 A.RNA1:M.DNA Marker,N.阴性对照,1.编号为24-1的番茄样品,2.编号为24-2的番茄样品,a.RNA1-Ⅰ-F/R扩增,b.RNA1-Ⅱ-F/R扩增,c.RNA1-Ⅲ-F/R扩增,d.RNA1-Ⅳ-F/R扩增,e.RNA1-Ⅴ-F/R扩增;B.RNA2:M.DNA Marker,N.阴性对照,1.编号为24-1的番茄样品,2.编号为24-2的番茄样品,f.RNA2-Ⅰ-F/R扩增,g.RNA2-Ⅱ-F/R扩增,h.RNA2-Ⅲ-F/R扩增,i.RNA2-Ⅳ-F/R扩增,j.RNA2-Ⅴ-F/R扩增。

Fig.2 RNA1 and RNA2 full-length amplification of ToCV-SJZ isolate A.RNA1:M.DNA Marker,N.Negative control,1.Tomato sample 24-1,2.Tomato sample 24-2,a.The detection results of RNA1-Ⅰ-F/R,b.The detection results of RNA1-Ⅱ-F/R,c.The detection results of RNA1-Ⅲ-F/R,d.The detection results of RNA1-Ⅳ-F/R,e.The detection results of RNA1-Ⅴ-F/R;B.RNA2:M.DNA Marker,N.Negative control,1.Tomato sample 24-1,2.Tomato sample 24-2,f.The detection results of RNA2-Ⅰ-F/R,g.The detection results of RNA2-Ⅱ-F/R,h.The detection results of RNA2-Ⅲ-F/R,i.The detection results of RNA2-Ⅳ-F/R,j.The detection results of RNA2-Ⅴ-F/R.

图3 ToCV分离物基因组结构

Fig.3 Genome structure of ToCV isolates

表3 ToCV-SJZ 分离物RNA1和RNA2的ORFs

Tab.3 RNA1 and RNA2 ORFs of ToCV-SJZ isolate

序列 Sequences	开放阅读框 ORFs	位置/nt Location	氨基酸序列长度/aa Amino acid sequence length	编码的蛋白 Protein
ToCV-SJZ-RNA1	ORF 1a	304~6 141	1 945	木瓜蛋白酶、甲基转移酶、斛旋酶
	ORF 1b	6 056~7 657	533	RdRp
	ORF 2	7 664~8 242	192	P22
	ORF 3	8 265~8 420	51	P5
ToCV-SJZ-RNA2	ORF 1	733~2 397	534	Hsp70h
	ORF 2	2 407~2 610	67	P8
	ORF 3	2 562~4 115	517	P59
	ORF 4	4 097~4 333	78	P9
	ORF 5	4 333~5 106	257	CP
	ORF 6	5 112~7 121	669	mCP
	ORF 7	7 125~7 823	232	P27
	ORF 8	7 832~8 029	65	P7

表4 ToCV-SJZ分离物与其他ToCV分离物的RNA1全长序列及ORFs的相似性比较

Tab.4 Homology comparison of full-length RNA1 sequence and ORFs between ToCV-SJZ and other isolates of ToCV%

分离物 Isolate	RNA1登录号 GenBank accession No.of RNA1	RNA1	ORF 1a	ORF 1b	ORF 2	ORF 3
ToCV-XS	KY618796.1	85.2	91.9	98.2	88.5	78.4
ToCV-AT80/99-IC	KJ740256.1	74.5	87.8	96.2	67.7	64.7
ToCV-BR	MT279194.1	74.5	87.5	96.4	66.7	64.7
ToCV-CQ630	PQ213439.1	77.3	87.6	96.4	65.3	64.7
ToCV-DSMZ PV-1242	ON398512.1	74.6	87.8	96.4	67.2	64.7
ToCV-FERA_160205	KY810786.1	78.7	87.7	96.4	66.7	64.7
ToCV-Florida	AY903447.1	78.6	87.5	96.4	66.7	64.7
ToCV-Kas	KY419526.1	78.5	87.4	96.4	65.6	64.7
ToCV-KGS	LC711108.1	78.3	87.7	96.2	65.6	64.7
ToCV-PAK1	MN869004.1	78.6	87.6	96.2	67.7	64.7
ToCV-SDSG	KC709509.1	78.6	87.7	96.4	66.1	64.7
ToCV-SDTA	OR246919.1	77.3	87.6	96.4	65.8	64.7
ToCR-186	KY471129.1	78.3	87.5	96.2	65.6	62.7
ToCV-BJ	KC887998.1	78.6	87.5	96.4	66.7	64.7
ToCV-PAP-JJ6	OR865222.1	77.2	87.6	96.4	66.3	64.7
ToCV-SH-SIA	MW490611.1	78.6	87.9	96.4	66.7	64.7

表5 ToCV-SJZ分离物与其他ToCV分离物的RNA2全长序列及ORFs的相似性比较

Tab.5 Homology comparison of full-length RNA2 sequence and ORFs between ToCV-SJZ and other isolates of ToCV %

分离物 Isolate	RNA2登录号 GenBank accession No.of RNA2	RNA2	ORF 1	ORF 2	ORF 3	ORF 4	ORF 5	ORF 6	ORF 7	ORF 8
ToCV-XS	KY618797.1	92.4	98.6	-	98.1	92.7	95.7	93.4	97.8	78.5
ToCV-AT80/99-IC	KJ740257.1	78.2	94.4	76.1	90.1	94.9	92.2	82.2	90.1	76.9
ToCV-BR	MT279195.1	77.7	94.4	80.6	90.7	96.2	91.8	82.2	90.5	76.9
ToCV-CQ630	PQ213440.1	73.7	93.9	77.6	89.9	96.2	92.2	82.5	89.2	75.4
ToCV-DSMZ PV-1242	ON398513.1	78.2	94.9	79.1	90.3	96.2	91.4	82.4	89.7	76.9
ToCV-FERA_160205	KY810787.1	78.2	94.6	77.6	89.9	96.2	92.2	82.4	89.7	76.9
ToCV-Florida	AY903448.1	78.1	94.2	79.1	90.3	96.2	92.2	82.7	89.7	76.9
ToCV-Kas	KY419528.1	78.1	94.2	79.1	90.7	94.9	91.1	82.7	89.7	76.9
ToCV-KGS	LC711109.1	77.3	94.4	79.1	90.7	96.2	92.2	82.8	89.2	76.9
ToCV-PAK1	MN869006.1	78.1	94.6	79.1	90.3	96.2	91.4	82.5	90.1	76.9
ToCV-SDSG	KC709510.1	78.3	94.6	80.6	90.5	94.9	92.0	82.7	89.7	76.9
ToCV-SDTA	OR246920.1	82.6	94.6	79.1	90.7	96.2	96.1	92.2	98.7	76.9
ToCR-186	KY471130.1	78.3	94.8	77.6	90.9	96.2	92.2	81.5	90.1	76.9
ToCV-BJ	KC887999.1	78.2	94.0	80.6	90.1	93.6	92.2	82.4	89.7	76.9
ToCV-PAP-JJ6	OR865223.1	76.8	94.6	79.1	90.7	96.2	92.6	82.8	89.7	76.9
ToCV-SH-SIA	MW490612.1	77.9	94.9	78.7	90.3	96.2	91.4	82.2	89.2	76.9

图4 ToCV-SJZ和其他ToCV分离物的RNA1和RNA2系统进化 ToCV(ToCR).番茄褪绿病毒;SPCSV.甘薯褪绿矮化病毒;DVCV.双角草脉褪绿病毒;SPaV.草莓白化相关病毒;BYVaV.黑莓黄脉相关病毒;CCYV.瓜类褪绿黄化病毒;LCV.莴苣褪绿病毒;CYSDV.瓜类黄色矮化失调病毒;TVCV.北美血根草脉褪绿病毒;LIYV.莴苣侵染性黄化病毒;TICV.番茄侵染性褪绿病毒。图5同。A.基于RNA1全长序列构建的系统进化树;B.基于RNA2全长序列构建的系统进化树。

Fig.4 Phylogenetic analysis of RNA1 and RNA2 sequences of ToCV-SJZ and other ToCV isolates ToCV(ToCR).Tomato chlorosis virus;SPCSV.Sweet potato chlorotic stunt virus;DVCV.Diodia vein chlorosis virus;SPaV.Strawberry pallidosis associated virus;BYVaV.Blackberry yellow vein-associated virus;CCYV.Cucurbit chlorotic yellows virus;LCV.Lettuce chlorosis virus;CYSDV.Cucurbit yellow stunting disorder virus;TVCV.Tetterwort vein chlorosis virus;LIYV.Lettuce infectious yellows virus;TICV.Tomato infectious chlorosis virus.The same as Fig.5.A.Phylogenetic analysis based on full-length RNA1 sequence;B.Phylogenetic analysis based on full-length RNA2 sequence.

图5 ToCV-SJZ和其他ToCV分离物的Hsp70h和CP蛋白系统进化 A.基于Hsp70h蛋白序列构建的系统进化树;B.基于CP蛋白序列构建的系统进化树。

Fig.5 Phylogenetic analysis of Hsp70h and CP proteins of ToCV-SJZ and other ToCV isolates A.Phylogenetic analysis based on Hsp70h protein sequence;B.Phylogenetic analysis based on CP protein sequence.

图6 SJZ-CP-216-F/R、Flo-CP-250-F/R和ToCV-CP-154-F/R特异性检测 M.DNA Marker;N.阴性对照;1-2.ToCV-SJZ;3-4.ToCV-Florida。

Fig.6 Primer specific testing of SJZ-CP-216-F/R, Flo-CP-250-F/R and ToCV-CP-154-F/R M.DNA Marker;N.Negative control;1-2.ToCV-SJZ;3-4.ToCV-Florida.

图7 RT-qPCR扩增曲线和标准曲线 A.SJZ-CP-216-BS的RT-qPCR扩增曲线:a1~a5.拷贝数3.58×10⁷~3.58×10³拷贝/μL的SJZ-CP-216-BS质粒样品;B.SJZ-CP-216-BS的RT-qPCR熔解曲线;C.SJZ-CP-216-BS的标准曲线;D.Flo-CP-250-BS的RT-qPCR扩增曲线:c1~c5.拷贝数3.68×10⁷~3.68×10³拷贝/μL的Flo-CP-250-BS质粒样品;E.Flo-CP-250-BS的RT-qPCR熔解曲线;F.Flo-CP-250-BS的标准曲线;G.ToCV-CP-154-SJZ-BS的RT-qPCR扩增曲线:e1~e5.拷贝数4.23×10⁷~4.23×10³拷贝/μL的ToCV-CP-154-SJZ-BS质粒样品;H.ToCV-CP-154-SJZ-BS的RT-qPCR熔解曲线;I.ToCV-CP-154-SJZ-BS的标准曲线;J.ToCV-CP-154-Flo-BS的RT-qPCR扩增曲线:g1~g5.拷贝数3.86×10⁷~3.86×10³拷贝/μL的ToCV-CP-154-Flo-BS质粒样品;K.ToCV-CP-154-Flo-BS的RT-qPCR熔解曲线;L.ToCV-CP-154-Flo-BS的标准曲线。

Fig.7 RT-qPCR amplification curve and standard curve A.RT-qPCR amplification curve of SJZ-CP-216-BS:a1-a5.SJZ-CP-216-BS plasmid with 3.58×10⁷-3.58×10³copies/μL;B.RT-qPCR melting curve of SJZ-CP-216-BS;C.Standard curve of SJZ-CP-216-BS;D.RT-qPCR amplification curve of Flo-CP-250-BS:c1-c5.Flo-CP-250-BS plasmid with 3.68×10⁷-3.68×10³copies/μL;E.RT-qPCR melting curve of Flo-CP-250-BS;F.Standard curve of Flo-CP-250-BS;G.RT-qPCR amplification curve of ToCV-CP-154-SJZ-BS:e1-e5.ToCV-CP-154-SJZ-BS plasmid with 4.23×10⁷-4.23×10³copies/μL;H.RT-qPCR melting curve of ToCV-CP-154-SJZ-BS;I.Standard curve of ToCV-CP-154-SJZ-BS;J.RT-qPCR amplification curve of ToCV-CP-154-Flo-BS:g1-g5.ToCV-CP-154-Flo-BS plasmid with 3.86×10⁷-3.86×10³copies/μL;K.RT-qPCR melting curve of ToCV-CP-154-Flo-BS;L.Standard curve of ToCV-CP-154-Flo-BS.

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