Studies on Detection of Cucumis Melo Endornavirus Using TaqMan RT-qPCR Method

doi:10.16420/j.issn.0513-353x.2021-0620

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (11): 2471-2478.doi: 10.16420/j.issn.0513-353x.2021-0620

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Studies on Detection of Cucumis Melo Endornavirus Using TaqMan RT-qPCR Method

ZHAO Liqun¹, ZHANG Xiaofei², QIU Yanhong²^,³^,^*(), LIU Hui², ZHANG Jian²^,³, YANG Jingjing², ZHANG Haijun²^,³, XU Xiulan²^,³, WEN Changlong²^,³^,^*()

¹Beijing Agricultural Extension Station，Beijing 100029，China
²Key Laboratory of Biology and Genetics Improvement of Horticultural Crops（North China），Institute of Vegetable Science，Beijing Academy of Agricultural and Forestry Sciences，Beijing 100097，China
³Supervision，Inspection and Test Center of Vegetable Seed Quality of Ministry of Agriculture and Rural Affairs，Beijing 100097，China

Received:2022-01-19 Revised:2022-03-24 Online:2022-11-25 Published:2022-11-25
Contact: QIU Yanhong,WEN Changlong E-mail:qiuyanhong@nercv.org;wenchanglong@nercv.org

Abstract

Abstract:

Cucumis melo endornavirus is an important plant virus that infects a wide range of hosts，occurs popularly and transmits highly in seeds. One set of specific primers and probes were designed according to the conserved sequence of CmEV genome，and a detecting method of CmEV was established using the Real-Time Fluorescent Quantitative PCR with TaqMan probes（TaqMan RT-qPCR）. This detection method could detect the virus in 1 × 10^-2ng · μL^-1 RNA samples and 1.8 × 10³ copies · μL^-1 plasmids，the sensitivity of which was about one hundred times higher than traditional RT-PCR. We used the CmEV TaqMan RT-qPCR technique to detect 11 seedling lots of Cucumis melo，Citrullus lanatus，Cucumis sativus，Cucurbita moschata stock that randomly sampled from the main cucurbit industries of Beijing，Shandong，Liaoning and Hainan provinces. It was found that Cucumis melo seedling from Shandong and Hainan industries carried CmEV. The novel TaqMan RT-qPCR method was proved to be efficient in testing CmEV，which could be a technical support for precisely detecting and scientific controlling of this virus，and could help preventing the virus spread from the source.

Key words: cucumis melo endornavirus, TaqMan qPCR, cucurbits crops, seedling-borne disease

CLC Number:

S652

ZHAO Liqun, ZHANG Xiaofei, QIU Yanhong, LIU Hui, ZHANG Jian, YANG Jingjing, ZHANG Haijun, XU Xiulan, WEN Changlong. Studies on Detection of Cucumis Melo Endornavirus Using TaqMan RT-qPCR Method[J]. Acta Horticulturae Sinica, 2022, 49(11): 2471-2478.

Figures/Tables 7

Table 1 The information of Cucurbits crops seedlings for detection of CmEV

序号 Number	种苗 Seedling	产地 Area	序号 Number	瓜类种苗 Seedling	产地 Area
1	甜瓜 Melon	北京 Beijing	7	黄瓜Cucumber	北京 Beijing
2	甜瓜 Melon	山东 Shandong	8	黄瓜Cucumber	山东 Shandong
3	甜瓜 Melon	辽宁 Liaoning	9	黄瓜Cucumber	辽宁 Liaoning
4	甜瓜 Melon	海南 Hainan	10	南瓜砧木Pumpkin stock	北京 Beijing
5	西瓜 Watermelon	北京 Beijing	11	南瓜砧木Pumpkin stock	辽宁 Liaoning
6	西瓜 Watermelon	山东Shandong

Table 2 The sequence of primers used for detection of the CmEV

引物 Primer	序列（5′-3′） Sequence	扩增片段/bp Amplified fragement
CmEV-qRT	P：CATACGCTTCGAAGACCCTCTTGCT	160
	F：GAGATACTTGTGGGTCAGTGGCAG
	R：GTCGGAAATTGGTTTATGTGGTG
CmEV-500	F：GGTAATGGCTTCATTCCCCATTAC	500
CmEV-RT	F：CGCGGAAATTGCACTTCCGAGAT	755
CmEV-RT	R：CTAGCAACTTGTTTGCTGGCACATG	755

Fig. 1 Specificity of TaqMan-based RT-qPCR for detection CmEV

Fig. 2 The amplified results of RT-PCR detection

Fig. 3 The detection sensitivity of TaqMan RT-qPCR and conventional RT-PCR of CmEV A：Amplification curves of TaqMan RT-qPCR of seven serial dilutions，B：TaqMan RT-qPCR standard quantification curve，C：Sensitivity of the conventional PCR assay.

Fig. 4 The detection sensitivity of TaqMan qPCR and conventional RT-PCR of CmEV A：Amplification curves of TaqMan qPCR of seven ten-fold serial dilutions，B：TaqMan qPCR standard quantification curve，C：Sensitivity of the conventional PCR assay.

Fig. 5 Detection of CmEV in major cucurbits crops seedlings

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Studies on Detection of Cucumis Melo Endornavirus Using TaqMan RT-qPCR Method

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