广东省蒲瓜病毒种类鉴定及多重RT-PCR检测方法的建立

doi:10.3864/j.issn.0578-1752.2023.23.010

中国农业科学 ›› 2023, Vol. 56 ›› Issue (23): 4684-4695.doi: 10.3864/j.issn.0578-1752.2023.23.010

广东省蒲瓜病毒种类鉴定及多重RT-PCR检测方法的建立

苏琦¹^,²(), 汤亚飞², 佘小漫², 蓝国兵², 于琳², 吴正伟¹, 李正刚²(), 何自福²()

¹ 广东海洋大学滨海农业学院，广东湛江 524088
² 广东省农业科学院植物保护研究所/广东省植物保护新技术重点实验室，广州 510640

收稿日期:2023-08-04 接受日期:2023-08-27 出版日期:2023-12-04 发布日期:2023-12-04
通信作者:
何自福，E-mail：hezf@gdppri.com。
李正刚，E-mail：lizhenggang@gdppri.com
联系方式: 苏琦，E-mail：sq1827266@163.com。
基金资助:
国家自然科学基金(32272509); 广东省农业科学院农业优势产业学科团队建设项目(202103TD); 广东省农业科学院农业优势产业学科团队建设项目(202105TD); 广州市科技计划(202102020504); 广州市青年科技人才托举项目(QT-2023-040); 广东省农业科学院科技人才培养专项(R2023PY-JX011); 广东省农业科学院协同创新中心项目(XT202210)

Identification of Viruses Infecting Bottle Gourd in Guangdong Province and Establishment of Multiplex RT-PCR Detection Method

SU Qi¹^,²(), TANG YaFei², SHE XiaoMan², LAN GuoBing², YU Lin², WU ZhengWei¹, LI ZhengGang²(), HE ZiFu²()

¹ College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524088, Guangdong
² Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences/Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangzhou 510640

Received:2023-08-04 Accepted:2023-08-27 Published:2023-12-04 Online:2023-12-04

摘要/Abstract

摘要：

【目的】探明侵染广东省蒲瓜的病毒种类，建立一种可以同时检测多种蒲瓜病毒的RT-PCR检测方法，提高蒲瓜病毒种类鉴定效率，为蒲瓜病毒病的精准监测与防控提供依据。【方法】2018—2022年间，从广东省广州市、惠州市、清远市、汕头市和湛江市的蒲瓜主要种植区采集蒲瓜病毒病疑似样品78份，对采集的样品按照地区和症状进行分类，提取每份样品的总RNA，将一个地区具有相同症状样品的RNA等量混合后进行小RNA深度测序分析。根据小RNA深度测序分析结果，设计特异引物进行RT-PCR验证，从而明确侵染广东省蒲瓜的病毒种类。挑选6种检出率高、危害严重的蒲瓜病毒，根据GenBank数据库设计多重PCR检测引物，通过优化反应体系和反应条件，建立同时检测6种蒲瓜病毒的多重RT-PCR方法。【结果】从采集的78份蒲瓜疑似病毒病样品中，共检测到12种病毒，按照检出率从高到低分别为葫芦内源RNA病毒（Lagenaria siceraria alphaendornavirus，LSEV）（64.1%）、黄瓜绿斑驳花叶病毒（cucumber green mottle mosaic virus，CGMMV）（62.8%）、小西葫芦虎纹花叶病毒（zucchini tigre mosaic virus，ZTMV）（51.3%）、西瓜绿斑驳花叶病毒（watermelon green mottle mosaic virus，WGMMV）（43.6%）、西瓜病毒A（watermelon virus A，WVA）（32.1%）、番木瓜环斑病毒（papaya ringspot virus，PRSV）（19.2%）、小西葫芦黄花叶病毒（zucchini yellow mosaic virus，ZYMV）（9.0%）、瓜类蚜传黄化病毒（cucurbit aphid-borne yellows virus，CABYV）（7.7%）、中国南瓜曲叶病毒（squash leaf curl China virus，SLCCNV）（7.7%）、瓜类褪绿黄化病毒（cucurbit chlorotic yellows virus，CCYV）（5.1%）、瓜类黄矮失调病毒（cucurbit yellow stunting disorder virus，CYSDV）（3.8%）、甜瓜黄斑病毒（melon yellow spot virus，MYSV）（1.3%）。78份样品中，复合侵染率高达80.8%，其中2、3、4、5、6和7种病毒复合侵染的检出率分别为14.1%、16.7%、23.1%、17.9%、7.7%和1.3%。在多重RT-PCR体系中先加入WVA+CCYV+ZTMV+PRSV引物，12个反应后再加入CGMMV+WGMMV引物，引物体积依次为WVA 0.5 μL、CCYV 0.5 μL、CGMMV 0.4 μL、WGMMV 0.4 μL、ZTMV 0.6 μL、PRSV 0.6 μL，从而确立了一种同时扩增6种病毒的多重RT-PCR检测方法。【结论】危害广东省蒲瓜的主要病毒有12种，其中CGMMV、ZTMV、WGMMV、WVA为优势病毒；复合侵染现象较普遍，以3、4和5种病毒的复合侵染形式占比较高；研发出一种同时检测蒲瓜上6种病毒的多重RT-PCR检测技术，提高了蒲瓜病毒种类鉴定效率。

关键词: 蒲瓜, 病毒病, 小RNA深度测序, RT-PCR检测, 多重PCR方法

Abstract:

【Objective】The objective of this research is to determine the various viruses that infect bottle gourd, establish a multiplex RT-PCR detection method that can simultaneously detect multiple bottle gourd viruses, so as to enhance the efficiency of identifying bottle gourd virus species, and provide a foundation for accurate prevention and control of bottle gourd viral diseases.【Method】From 2018 to 2022, 78 bottle gourd samples of suspected virus infection were collected from the main planting areas of bottle gourd in Guangdong Province, including Guangzhou, Huizhou, Qingyuan, Shantou, and Zhanjiang. The collected samples were sorted by region and symptom, and the total RNA of each sample was extracted. The RNA samples with similar symptoms from each region were mixed for small RNA deep sequencing analysis. According to the results of small RNA deep sequencing analysis, specific primers were designed for RT-PCR verification to identify the specific virus species infecting bottle gourd in Guangdong Province. Six viruses with higher incidence and more losses were selected, and multiplex PCR detection primers based on the GenBank database were designed. After optimizing the reaction system and conditions, a method for simultaneous detection of all six viruses via multiplex RT-PCR was established.【Result】A total of 12 viruses were detected in 78 bottle gourd samples of suspected virus infection. These viruses were ranked in order of infection rate, from highest to lowest: Lagenaria siceraria alphaendornavirus (LSEV) (64.1%), cucumber green mottle mosaic virus (CGMMV) (62.8%), zucchini tigre mosaic virus (ZTMV) (51.3%), watermelon green mottle mosaic virus (WGMMV) (43.6%), watermelon virus A (WVA) (32.1%), papaya ringspot virus (PRSV) (19.2%), zucchini yellow mosaic virus (ZYMV) (9.0%), cucurbit aphid-borne yellows virus (CABYV) (7.7%), squash leaf curl China virus (SLCCNV) (7.7%), cucurbit chlorotic yellows virus (CCYV) (5.1%), cucurbit yellow stunting disorder virus (CYSDV) (3.8%), melon yellow spot virus (MYSV) (1.3%). It was found that 80.8% of the 78 samples were infected with multiple viruses. The types of mixed infection included 2 viruses (14.1%), 3 viruses (16.7%), 4 viruses (23.1%), 5 viruses (17.9%), 6 viruses (7.7%) and 7 viruses (1.3%), respectively. The WVA+CCYV+ZTMV+PRSV primers were added firstly in the multiplex RT-PCR system, followed by the WGMMV+ CGMMV primers after 12 reaction cycles. The volumes of primers were used: WVA 0.5 μL, CCYV 0.5 μL, CGMMV 0.4 μL, WGMMV 0.4 μL, ZTMV 0.6 μL, and PRSV 0.6 μL. Thus, a multiplex RT-PCR assay capable of simultaneously amplifying six viruses was established.【Conclusion】There are 12 primary viruses that infect bottle gourd in Guangdong Province. Among them, CGMMV, ZTMV, WGMMV, and WVA are the most predominant viruses. Mixed infection is a commonly observed phenomenon, with three, four, and five viruses’ co-infection being the most prevalent types. A multiplex RT-PCR detection method has been developed to simultaneously identify six viruses in bottle gourd, which improves the detection efficiency of virus species in bottle gourd.

Key words: bottle gourd, virus disease, small RNA deep sequencing, RT-PCR detection, multiplex PCR method

苏琦, 汤亚飞, 佘小漫, 蓝国兵, 于琳, 吴正伟, 李正刚, 何自福. 广东省蒲瓜病毒种类鉴定及多重RT-PCR检测方法的建立[J]. 中国农业科学, 2023, 56(23): 4684-4695.

SU Qi, TANG YaFei, SHE XiaoMan, LAN GuoBing, YU Lin, WU ZhengWei, LI ZhengGang, HE ZiFu. Identification of Viruses Infecting Bottle Gourd in Guangdong Province and Establishment of Multiplex RT-PCR Detection Method[J]. Scientia Agricultura Sinica, 2023, 56(23): 4684-4695.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.23.010

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引物Primer	序列Sequence	扩增长度Amplification length (bp)	Tm (℃)
WGMMV-F	TGACCAGTCTATTCTGGACGT	1200	55
WGMMV-R	TAAGGCAGCAATTTCGAGAGT	1200	55
CCYV-F	GCCATAACCATTACGGGAGA	389	55
CCYV-R	CGCAGTGAAAAACCCAAACT	389	55
CGMMV-F	AGACGCTCATTTACTATCAACG	1080	55
CGMMV-R	AGACTCGACTACAGAAGCTC	1080	55
CYSDV-F	TGTTTATGAAATCCGTTTGTGATAGTCT	853	55
CYSDV-R	GACTGCATATTAGCACTTATTAACCA	853	55
LSEV-F	ATTCCTGGGCAACATTTAATGAT	1280	55
LSEV-R	TACCTGGAATTCTGCAACCT	1280	55
MYSV-F	GAAAATCCAAGAACTTCTTAGTGGTG	775	55
MYSV-R	CTTTACTCTTAGAAGAGCTCTCACCC	775	55
CABYV-F	ACACCCAGTACATCTGGGAA	888	55
CABYV-R	CAAAACCGGAGCAGTCAG	888	55
PRSV-F	TCAACGTCGGAACTAGTGGAAC	700	55
PRSV-R	ATTACGCATACCCAGGAGAGAG	700	55
SLCCNV-F	ACAACATGTGGGATCCACTTAT	548	60
SLCCNV-R	GTCTTGATATTTTCGTCCATCCAT	548	60
WVA-F	AATGGGAGGCTCAATAAATGTATT	1350	55
WVA-R	GCATTCCTGCATCCTTCATT	1350	55
ZTMV-F	ACTGTTCCTCGAATTAAAACATTTACTGA	660	55
ZTMV-R	TCCAAGGAGAGAGTGCATGTC	660	55
ZYMV-F	ACTCAGAATCTCAAGCTCTCAT	1153	55
ZYMV-R	AGCAATAATATGTATGGGTCATCG	1153	55

引物Primer	序列Sequence	扩增长度Amplification length (bp)	体积<BOLD>V</BOLD>olume (μL)	Tm (℃)
WVA-F	AATGGGAGGCTCAATAAATGTATT	1514	0.5	55
WVA-R	CTCTCCTCCATTCATATCTGCT	1514	0.5	55
CCYV-F	ACAACAAATCCTTAGGGTGTCTG	750	0.5	55
CCYV-R	CGAATACAAACAATAACGCGTCCA	750	0.5	55
CGMMV-F	GTATTCGATGCAGTTACAAGTATAATAGC	600	0.4	55
CGMMV-R	CGCTGAGGTTGGCTGACA	600	0.4	55
WGMMV-F	GTCGCACAGAGACGAATTAGT	452	0.4	55
WGMMV-R	AATCGCGACGAATCTCT	452	0.4	55
ZTMV-F	AATCAAAGGATGGCGCCGT	344	0.6	55
ZTMV-R	GTGGCACGCGTGTTTGA	344	0.6	55
PRSV-F	CAGCCAATCAAAGCTGGGG	212	0.6	55
PRSV-R	GCCTTCGCTTGCTTCAATTGTA	212	0.6	55

病毒名称<BOLD>V</BOLD>irus name	科Family	属Genus
瓜类褪绿黄化病毒Cucurbit chlorotic yellows virus (CCYV)	长线形病毒科Closteroviridae	毛形病毒属Crinivirus
黄瓜绿斑驳花叶病毒Cucumber green mottle mosaic virus (CGMMV)	杆状病毒科Virgaviridae	烟草花叶病毒属Tobamovirus
葫芦内源RNA病毒Lagenaria siceraria alphaendornavirus (LSEV)	内生病毒科Endornaviridae	甲型内源病毒属Alphaendornavirus
甜瓜黄斑病毒Melon yellow spot virus (MYSV)	番茄斑萎病毒科Tospoviridae	正番茄斑萎病毒属Orthotospovirus
番木瓜环斑病毒Papaya ringspot virus (PRSV)	马铃薯Y病毒科Potyviridae	马铃薯Y病毒属Potyvirus
中国南瓜曲叶病毒Squash leaf curl China virus (SLCCNV)	双生病毒科Geminiviridae	菜豆金黄花叶病毒属Begomovirus
西瓜病毒A Watermelon virus A (WVA)	乙型线状病毒科Betaflexiviridae	西瓜病毒A属Wamavirus
西瓜绿斑驳花叶病毒Watermelon green mottle mosaic virus (WGMMV)	杆状病毒科Virgaviridae	烟草花叶病毒属Tobamovirus
小西葫芦虎纹花叶病毒Zucchini tigre mosaic virus (ZTMV)	马铃薯Y病毒科Potyviridae	马铃薯Y病毒属Potyvirus
瓜类黄矮失调病毒Cucurbit yellow stunting disorder virus (CYSDV)	长线形病毒科Closteroviridae	毛形病毒属Crinivirus
瓜类蚜传黄化病毒Cucurbit aphid-borne yellows virus (CABYV)	黄症病毒科Luteoviridae	马铃薯卷叶病毒属Polerovirus
小西葫芦黄花叶病毒Zucchini yellow mosaic virus (ZYMV)	马铃薯Y病毒科Potyviridae	马铃薯Y病毒属Potyvirus

	复合侵染类型<BOLD>M</BOLD>ixed infection type	感染病样数Number of infected samples
2种病毒 Two viruses	CGMMV+WVA	5
	CGMMV+WGMMV	2
	CGMMV+ZTMV	1
	LSEV+ZTMV	1
	WVA+PRSV	1
	WGMMV+LSEV	1
3种病毒 Three viruses	LSEV+ZTMV+ZYMV	1
	CGMMV+ ZTMV+ZYMV	1
	CGMMV+PRSV+WGMMV	1
	CGMMV+WGMMV+WVA	1
	CGMMV+LSEV+WGMMV	1
	CGMMV+ LSEV+ZTMV	2
	LSEV+ZTMV+WVA	1
	LSEV+WGMMV+CABYV	1
	LSEV+SLCCNV+WGMMV	1
	LSEV+WGMMV+ZTMV	2
	LSEV+ZTMV+PRSV	1
4种病毒 Four viruses	LSEV+ZYMV+ZTMV+WVA	1
	CGMMV+MYSV+PRSV+WGMMV	1
	CGMMV+WVA+LSEV+WGMMV	4
	ZTMV+LSEV+PRSV+CGMMV	1
	WGMMV+CGMMV+LSEV+ZTMV	6
	WGMMV+WVA+LSEV+ZTMV	1
	ZTMV+LSEV+CGMMV+WVA	1
	LSEV+SLCCNV+WGMMV+PRSV	1
	LSEV+SLCCNV+WGMMV+ZTMV	1
	LSEV+WGMMV+ZTMV+PRSV	1
5种病毒 Five viruses	CGMMV+WVA+LSEV+ZTMV+WGMMV	5
	ZTMV+LSEV+PRSV+WGMMV+CGMMV	2
	ZTMV+ZYMV+WGMMV+LSEV+PRSV	1
	ZTMV+ZYMV+WGMMV+LSEV+CGMMV	1
	CGMMV+LSEV+WGMMV+ZTMV+CABYV	2
	LSEV+SLCCNV+WGMMV+ZTMV+PRSV	1
	LSEV+WGMMV+WVA+ZTMV+PRSV	1
	LSEV+SLCCNV+WGMMV+WVA+PRSV	1
6种病毒 Six viruses	WVA+ZYMV+LSEV+CGMMV+WGMMV+ZTMV	1
	ZTMV+ZYMV+WGMMV+LSEV+CGMMV+PRSV	1
	CCYV+CGMMV+LSEV+WGMMV+ZTMV+CYSDV	1
	CGMMV+LSEV+SLCCNV+WGMMV+ZTMV+CABYV	1
	CCYV+CGMMV+LSEV+WGMMV+CABYV+CYSDV	1
	CCYV+LSEV+WGMMV+WVA+ZTMV+PRSV	1
7种病毒 Seven viruses	CCYV+CGMMV+LSEV+WGMMV+ZTMV+CABYV+CYSDV	1

广东省蒲瓜病毒种类鉴定及多重RT-PCR检测方法的建立

Identification of Viruses Infecting Bottle Gourd in Guangdong Province and Establishment of Multiplex RT-PCR Detection Method

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