Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (20): 4337-4347.doi: 10.3864/j.issn.0578-1752.2021.20.008

• PLANT PROTECTION • Previous Articles     Next Articles

The Detection of Citrullus lanatus Cryptic Virus Using TaqMan-qPCR Method

ZHAO LiQun1(),QIU YanHong2,3(),ZHANG XiaoFei2,LIU Hui2,YANG JingJing2,ZHANG Jian2,3,ZHANG HaiJun2,3,XU XiuLan2,3,WEN ChangLong2,3()   

  1. 1Beijing Agricultural Extension Station, Beijing 100029
    2Beijing Vegetable Research Center (BVRC), Beijing Academy of Agricultural and Forestry Sciences/National Engineering Research Center for Vegetables, Beijing 100097
    3Supervision, Inspection and Test Center of Vegetable Seed Quality of Ministry of Agriculture and Rural Affairs, Beijing 100097
  • Received:2021-03-01 Accepted:2021-04-09 Online:2021-10-16 Published:2021-10-25
  • Contact: YanHong QIU,ChangLong WEN E-mail:zhaoliqun2009@163.com;qiuyanhong@nercv.org;wenchanglong@nercv.org

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Abstract:

【Objective】Citrullus lanatus cryptic virus (CiLCV) is an important seed-transmitted virus that newly-emerging in watermelon in recent years. The objective of this study is to develop a detection method for CiLCV with real-time fluorescent quantitative PCR with TaqMan probes (TaqMan-qPCR), and to provide technical supports for the CiLCV detection from seeds and seedlings, and also for disease controlling in the future. 【Method】The CiLCV was found in Beijing watermelon producing area with high-throughput sequencing based on small RNAs. The full sequence of CiLCV-dsRNA1 and CiLCV-dsRNA2 was cloned and specific amplifying primers were designed to set up the detection method of the RT-PCR and TaqMan-qPCR. The other eight common viruses (cucumber green mottled mosaic virus, cucumber mosaic virus, Cucumis melo endornavirus, cucurbit chlorotic yellows virus, squash mosaic virus, tomato spotted wilt virus, watermelon mosaic virus, zucchini yellow mosaic virus) were used as control to analyze the method specificity. The standard curve of CiLCV was performed to evaluate the method sensitivity. Furthermore, the novel detection methods of TaqMan-qPCR and RT-PCR were used to inspect CiLCV in watermelon, cucumber, melon, and rootstock pumpkin seedlings that randomly collected from the main producing areas of cucurbits crop in North China. 【Result】The small RNAs data with high-quality were obtained, and 17 assembled contigs were found to share homology with CiLCV genomes after data analysis. The full-length of CiLCV-dsRNA1 and CiLCV-dsRNA2 was cloned with 1 603 and 1 466 nt in length, respectively, sharing the highest nt sequence identity (about 99.4% and 99.8%, respectively) with CiLCV that isolated from Henan Province (GenBank number KY081285, KY081284). The established RT-PCR for CiLCV showed a single amplified band, while the novel TaqMan-qPCR for detecting CiLCV showed good sensitivity and specificity, and could detect about 2×103 copies of CiLCV. The detection sensitivity of TaqMan-qPCR was about 100 times higher than that of RT-PCR. In addition, only one Beijing-watermelon sample was CiLCV positive, while the rest watermelon, melon, cucumber, and rootstock pumpkin seedlings were CiLCV negative, indicating that the virulence of seedlings in the main producing areas of cucurbits crop in China was not high. 【Conclusion】The established TaqMan-qPCR method for CiLCV detection has high specificity and sensitivity, and is suitable for rapid detection and accurate identification of CiLCV at ports and laboratories. In view of CiLCV can spread rapidly through seeds and seedlings, China should pay more attention to the detection of virulent seeds and seedlings to prevent the virulent seeds and seedlings flowing into the production process and then being transported and spread to the whole country, which may cause great economic losses to the industry.

Key words: Citrullus lanatus cryptic virus (CiLCV), real-time qPCR, TaqMan probe, cucurbits crop, seedling-borne disease

Table 1

The information of the main cucurbits crop seedlings for detection"

序号
Series		 葫芦科作物种苗
Cucurbits seedling		 来源
Origin		 序号
Series		 葫芦科作物种苗
Cucurbits seedling		 来源
Origin
1 西瓜种苗Watermelon seedling 北京Beijing 7 黄瓜种苗Cucumber seedling 辽宁Liaoning
2 西瓜种苗Watermelon seedling 北京Beijing 8 甜瓜种苗Melon seedling 北京Beijing
3 西瓜种苗Watermelon seedling 北京Beijing 9 甜瓜种苗Melon seedling 辽宁Liaoning
4 西瓜种苗Watermelon seedling 山东Shandong 10 南瓜砧木种苗Rootstock pumpkin seedling 北京Beijing
5 黄瓜种苗Cucumber seedling 北京Beijing 11 南瓜砧木种苗Rootstock pumpkin seedling 山东Shandong
6 黄瓜种苗Cucumber seedling 山东Shandong 12 南瓜砧木种苗Rootstock pumpkin seedling 辽宁Liaoning

Table 2

The primer sequences for CiLCV detection"

引物名称 Primer name 序列 Sequence (5′-3′) 目的 Purpose
CiLCV-R1-F AGAATTTTCCCCCAGTCAAC CiLCV-dsRNA1全长克隆
Full length cloning of CiLCV-dsRNA1
CiLCV-R1-R AGAAGGTAAGGGTTAAATAAC
CiLCV-R2-F AGAATTTTCCCCCAGTCAAC CiLCV-dsRNA2全长克隆
Full length cloning of CiLCV-dsRNA2
CiLCV-R2-R AGAAGGTAAGGGTTAAATAAC
CiCLV-588-F TCCAGACGTTGGCTACACAC RT-PCR检测CiLCV
CiLCV detection using RT-PCR
CiCLV-588-R ATTGCGAACCTCTCAGGTGG
DCiLCV-F1 AGAGGCACAGATGATGGATTTGG TaqMan-qPCR检测CiLCV
CiLCV detection using TaqMan-qPCR
DCiLCV-P1 AGCAGACCTTGAATTGATTACGGGTTC
DCiLCV-R1 CATCCAGAAAGGCACTGCTCAT
DCiLCV-F2 TGCCATTGAGACTGCTGTTCC TaqMan-qPCR检测CiLCV
CiLCV detection using TaqMan-qPCR
DCiLCV-P2 AGTACGACATGTATGGGGTCGCGC
DCiLCV-R2 TCTGGGTACACTAACTGTCGGATC

Fig. 1

The symptoms of watermelon infected with CiLCV and length distribution of small RNAs sequencing A、B/C are the symptoms of watermelon infected with CiLCV;D is the length distribution of small RNAs sequencing"

Fig. 2

The detection of CiLCV by RT-PCR"

Fig. 3

Analysis of specificity (A) and sensitivity (B) of detecting CiLCV with two sets of probes The blue and red curves indicate the amplification results of primer set 1 and primer set 2 for CiLCV, respectively, while the green curves indicate the amplification results of the other eight viruses"

Fig. 4

The analysis of detection sensitivity of TaqMan-qPCR (A) and establishment of standard curve (B) for CiLCV The template concentration of solution 1-7 is 11.8×100-11.8×10-6 ng·μL-1"

Fig. 5

The detection sensitivity of RT-PCR for CiLCV"

Fig. 6

The detection of CiLCV in main cucurbits crop seedlings with TaqMan-qPCR (A) and RT-PCR (B)"

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