Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (16): 3412-3420.doi: 10.3864/j.issn.0578-1752.2020.16.018

• RESEARCH NOTES • Previous Articles    

Multi-Gene-Based PCR Detection and Identification of Chilli veinal mottle virus

YANG HongKai1(),YANG JingWen1,SHEN JianGuo2(),CAI Wei3,GAO FangLuan1()   

  1. 1Fujian Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002
    2Technology Center of Fuzhou Customs District, Fuzhou 350001
    3Comprehensive Technical Service Center of Rongcheng Customs District, Fuqing 350300, Fujian
  • Received:2019-10-27 Accepted:2019-11-25 Online:2020-08-16 Published:2020-08-27
  • Contact: JianGuo SHEN,FangLuan GAO E-mail:yang_hong_kai@126.com;shenjg_agri@163.com;raindy@fafu.edu.cn

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Abstract:

【Objective】Chilli veinal mottle virus (ChiVMV), one of the most destructive pathogens causing server losses to chilli production, is an important plant virus in port quarantine. The objective of this study is to establish a fast and accurate multi-gene-based PCR detection method for ChiVMV.【Method】DAS-ELISA and RT-PCR were used to detect the infected chilli samples imported from India. Two pairs of specific primers were designed from the conserved regions of ChiVMV coat protein (CP) and cytoplasmic inclusion protein (CI), respectively. The multi-gene-based PCR detection method was established after optimizing parameters including primer dosage and annealing temperature. The established multi-gene-based PCR detection system was also used to detect chilli viruses including ChiVMV to verify the specificity of this system, and the different concentrations of cDNA in ChiVMV positive samples were amplified to determine its sensitivity. In addition, the utility of the system was tested by detecting ChiVMV in plant samples infected by this virus.【Result】All samples reacted positive on the DAS-ELISA test. RT-PCR amplifications of the ELISA-positive subsamples all generated expected fragments of 861 bp in size, using the specific primer pair of CP861-F/CP861-R. These results indicated that all samples were infected by ChiVMV. The specific target fragments of 337 and 655 bp were respectively amplified using the primer pairs of CP337-F/CP337-R and CI655-F/CI655-R in the multi-gene-based PCR detection system, whose optimized reaction system is cDNA 2 μL, CP337-F/CP337-R 0.625 μL (10 μmol·L-1), CI655-F/CI655-R 1.375 μL (10 μmol·L-1), 2×PCR Master Mix 12.5 μL, ddH2O 6.5 μL, annealing temperature 50℃, and for 35 cycles. The established multi-gene-based PCR detection system had good specificity and sensitivity, and two targeted fragments could be detected after the total DNA was diluted to 10-4. Other ChiVMV-infected samples were successfully detected by this method, generating two expected fragments of CP and CI, respectively.【Conclusion】The established multi-gene-based PCR detection method has strong specificity, high sensitivity, excellent repeatability, which is useful in the detection and identification of ChiVMV in port quarantine.

Key words: Chilli veinal mottle virus (ChiVMV), coat protein gene (CP), cytoplasmic inclusion protein gene (CI), multi-gene-based PCR detection

Table 1

Primers used for the detection of chilli samples in this study"

基因
Gene		 上下游引物序列
Upper and lower primer sequences (5′-3′)		 长度
Length (nt)		 退火温度
Tm (℃)		 目的片段
Expected size (bp)
CP CP861-F: GCAGGAGAGAGTGTTGATGC
CP861-R: CAATCCTCGAACGCCCAGCAG
CP337-F: ACACCTTCTTGATTATGCTCC
CP337-R: ATAAGGCTTCTCAGAATTGCG		 20
21
21
21		 59.9
63.9
56.1
56.1		 861a

337b
CI CI655-F: ACAGCRCCAGATGCAAAATC 20 58.0 655
CI655-R: CCTGTGCCYTGYGCATCMA 19 54.1

Table 2

Different gradient combinations and the primer dosage"

引物对
Primer pair		 不同引物用量梯度 Different gradient combinations (μL)
T1 T2 T3 T4 T5 T6 T7 T8 T9
CP337-F/CP337-R 1.5 1.375 1.25 1.125 1 0.875 0.75 0.625 0.5
CI655-F/CI655-R 0.5 0.625 0.75 0.875 1 1.125 1.25 1.375 1.5

Fig. 1

Conventional RT-PCR amplification of chilli from India Marker DNA (100 bp);1：PCR product of CP of ChiVMV isolate from India;2—4：Positive control (with known ChiVMV infected plant), negative control 1 (healthy plant) and negative control 2 (water), respectively"

Fig. 2

The results of RT-PCR by multi-gene-based PCR detection Marker DNA (100 bp);1：amplification of CP;2：amplification of CI;3：amplifications by multi-gene-based PCR detection;4：Negative control for amplification of CP (healthy plant);5：Negative control for amplification of CI (healthy plant);6：Negative control by multi-gene-based PCR detection (healthy plant)"

Fig. 3

Optimization of the annealing temperature in multi- gene-based PCR detection Marker DNA (100 bp);1—6：Annealing temperature of 46, 48, 50, 52, 54 and 56℃, respectively;7：Negative control (healthy plant)"

Fig. 4

Optimization of primer dosage in multi-gene-based PCR detection Marker DNA (100 bp);1：The combination of primer dosage T1 to T9, respectively;10：Negative control (healthy plant)"

Fig. 5

Specificity of the multi-gene-based PCR detection A：Multi-gene-based PCR detection;B：assay based on CP; C：assay based on CI。Marker DNA (100 bp);1：ChiVMV;2—5：Other main viruses of chilli;6：Negative control (healthy plant)"

Fig. 6

Sensitivity of the multi-gene-based PCR detection A：Multi-gene-based PCR detection;B：assay based on CP; C：assay based on CI。Marker DNA (100 bp);1—8：Dilution concentration of 100, 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, respectively;9：Negative control (healthy plant)"

Table 3

The chilli samples information for multi-gene-based PCR detection"

来源Origin 数量Quantity 阳性样品数量Number of positive samples 采集日期Date of collection
福州郊区Fuzhou suburb 5 5 2019-03-17
福清地区Fuqing area 25 0 2019-04-16

Fig. 7

PCR products of samples detected by multi-gene-based PCR Marker DNA (100 bp);1：Positive control;17：Negative control (healthy plant)。A：2-6 are chilli samples from Fuzhou suburb, 7-16 are chilli samples from Fuqing area;B：2-16 are chilli samples from Fuqing area。The same as Fig. 8"

Fig. 8

PCR products of samples detected by conventional RT-PCR"

[1] 孙国胜, 马志虎, 孙春青, 戴忠良, 毛忠良, 潘跃平. 早春辣椒连作免耕栽培技术. 中国瓜菜, 2015,28(1):55-56.
SUN G S, MA Z H, SUN Q C, DAI Z L, MAO Z L, PAN Y P. Early-season pepper continuous cropping and no-tillage cultivation technique. China Cucurbits and Vegetables, 2015,28(1):55-56. (in Chinese)
[2] 刘勇, 李凡, 李月月, 张松柏, 高希武, 谢艳, 燕飞, 张安盛, 戴良英, 程兆榜, 等. 侵染我国主要蔬菜作物的病毒种类、分布与发生趋势. 中国农业科学, 2019,52(2):239-261.
doi: 10.3864/j.issn.0578-1752.2019.02.005
LIU Y, LI F, LI Y Y, ZHANG S B, GAO X W, XIE Y, YAN F, ZHANG A S, DAI L Y, CHENG Z B, et al. Identification, distribution and occurrence of viruses in the main vegetables of China. Scientia Agricultura Sinica, 2019,52(2):239-261. (in Chinese)
doi: 10.3864/j.issn.0578-1752.2019.02.005
[3] 汤亚飞, 裴凡, 李正刚, 佘小漫, 于琳, 蓝国兵, 邓铭光, 何自福. 基于小RNA深度测序技术鉴定侵染广东辣椒的病毒种类. 中国农业科学, 2019,52(13):2256-2267.
TANG Y F, PEI F, LI Z G, SHE X M, YU L, LAN G B, DENG M G, HE Z F. Identification of viruses infecting peppers in Guangdong by small RNA deep sequencing. Scientia Agricultura Sinica, 2019,52(13):2256-2267. (in Chinese)
[4] ANINDYA R, JOSEPH J, GOWRI T D S, SAVITHRI H S. Complete genomic sequence of Pepper vein banding virus (PVBV): A distinct member of the genus Potyvirus. Archives of Virology, 2004,149(3):625-632.
pmid: 14991448
[5] RAVI K S, JOSEPH J, NAGARAJU N, PRASAD S K, REDDY H R, SAVITHRI H S. Characterization of a pepper vein banding virus from chilli pepper in India. Plant Disease, 1997,81(6):673-676.
pmid: 30861857
[6] ONG C A, VARGHESE G, TING W P. Aetiological investigations on a veinal mottle virus of chill (Capsicum annuum L.) newly recorded from peninsula Malaysia. Materials Research Bulletin, 1979,7:78-88.
[7] WANG J, LIU Z, NIU S, PENG M, WANG D, WENG Z, XIONG Z. Natural occurrence of Chilli veinal mottle virus on Capsicum chinense in China. Plant Disease, 2006,90(3):377.
pmid: 30786570
[8] 谭根堂, 史联联, 尚惠兰, 巩振辉. 陕西线辣椒病毒病病原检测简报. 辣椒杂志, 2003(3):32-33.
TAN G T, SHI L L, SHANG H L, GONG Z H. Diagnosis of viruses in chili pepper in Shaanxi Province. Journal of China Capsicum, 2003(3):32-33. (in Chinese)
[9] DING M, YANG C, ZHANG L, JIANG Z L, FANG Q, QIN X Y, ZHANG Z K. Occurrence of Chilli veinal mottle virus in Nicotiana tabacum in Yunnan, China. Plant Disease, 2011,95(3):357.
pmid: 30743508
[10] 刘健, 张德咏, 张松柏, 刘勇. 湖南和福建辣椒上辣椒脉斑驳病毒的检测及系统发育分析. 江苏农业科学, 2016,44(5):184-185.
LIU J, ZHANG D Y, ZHANG S B, LIU Y. Detection and phylogenetic analysis of Chilli veinal mottle virus in Hunan and Fujian. Jiangsu Agricultural Sciences, 2016,44(5):184-185. (in Chinese)
[11] 王达新, 王健华, 赵焕阁, 邱世明, 刘志昕. 辣椒叶脉斑驳病毒研究进展. 华南热带农业大学学报, 2007,13(2):32-36.
WANG D X, WANG J H, ZHAO H G, QIU S M, LIU Z X. Research progress in Chilli veinal mottle virus(ChiVMV). Journal of South China University of Tropical Agriculture, 2007,13(2):32-36. (in Chinese)
[12] ZHAO F F, XI D H, LIU J, DENG X G, LIN H H. First report of Chilli veinal mottle virus infecting tomato(Solanum lycopersicum) in China. Plant Disease, 2014,98(11):1589.
pmid: 30699808
[13] 张竹青. 辣椒病毒病研究[D]. 长沙: 湖南农业大学, 2009.
ZHANG Z Q. The research on Capsicum annumm L. virus diseases[D]. Changsha: Hunan Agricultural University, 2009. (in Chinese)
[14] 王少立, 谭玮萍, 杨园园, 代惠洁, 孙晓辉, 乔宁, 竺晓平. 山东省辣椒主要病毒种类的分子检测与鉴定. 中国农业科学, 2017,50(14):2728-2738.
WANG S L, TAN W P, YANG Y Y, DAI H J, SUN X H, QIAO N, ZHU X P. Molecular detection and identification of main viruses on pepper in Shandong Province. Scientia Agricultura Sinica, 2017,50(14):2728-2738. (in Chinese)
[15] SIRIWONG P, KITTIPAKORN K, IKEGAMI M. Characterization of Chilli vein-banding mottle virus isolated from pepper in Thailand. Plant Pathology, 1995,44(4):718-727.
doi: 10.1111/ppa.1995.44.issue-4
[16] MOURY B, PALLOIX A, CARANTA C, GOGNALONS P, SOUCHE S, SELASSIE K G, MARCHOUX G. Serological, molecular, and pathotype diversity of Pepper veinal mottle virus and Chili veinal mottle virus. Phytopathology, 2005,95(3):227-232.
pmid: 18943114
[17] 唐前君, 戴良英, 刘湘宁, 刘茂炎, 李迅, 刘双清, 李魏. 同时检测多种辣椒病毒的四重RT-PCR检测引物及方法: CN201510689135. 8[P]. (2015-12-23)[2019-10-27].
TANG Q J, DAI L Y, LIU X N, LIU M Y, LI X, LIU S Q, LI W. Simultaneous detection of multiple capsicum viruses by quadruple RT-PCR detection primers and methods: CN201510689135.8[P]. (2015-12-23)[2019-10-27]. (in Chinese)
[18] 汤亚飞, 何自福, 佘小漫, 蓝国兵. 一种检测辣椒脉斑驳病毒的RT-LAMP引物及试剂盒: CN201510527415.9[P]. (2015-11-11) [2019-10-27].
TANG Y F, HE Z F, SHE X M, LAN G B. RT-LAMP primer and kit for detecting Chilli veinal mottle virus: CN201510527415.9[P]. (2015-11-11)[2019-10-27]. (in Chinese)
[19] TSAI W S, HUANG Y C, ZHANG D Y, REDDY K, HIDAYAT S H, SRITHONGCHAI W, GREEN S K, JAN F J. Molecular characterization of the CP gene and 3' UTR of Chilli veinal mottle virus from South and Southeast Asia. Plant Pathology, 2008,57(3):408-416.
doi: 10.1111/j.1365-3059.2007.01780.x
[20] SCHAAD N W, FREDERICK R D. Real-time PCR and its application for rapid plant disease diagnostics. Canadian Journal of Plant Pathology, 2002,24(3):250-258.
[21] 谯天敏, 张静, 麻文建, 朱天辉. 双基因联合PCR检测水稻叶鞘网斑病菌. 南京农业大学学报, 2015,38(2):273-278.
QIAO T M, ZHANG J, MA W J, ZHU T H. The double gene-jointed PCR technique for detection of Cylindrocladium scoparium on rice. Journal of Nanjing Agricultural University, 2015,38(2):273-278. (in Chinese)
[22] 刘真真, 范昕建, 吴疆, 高燕渝. MecA、femB基因PCR联合扩增法检测耐甲氧西林金黄色葡萄球菌. 西部医学, 2011,23(4):604-605.
LIU Z Z, FAN X J, WU J, GAO Y Y. Evaluation of the mecA and femB duplex polymerase chain reaction for detection of methicillin- resistant Staphylococcus aureus. Medical Journal of West China, 2011,23(4):604-605. (in Chinese)
[23] ADAMS M J, ANTONIW J F, FAUQUET C M. Molecular criteria for genus and species discrimination within the familyPotyviridae. Archives of Virology, 2005,150(3):459-479.
pmid: 15592889
[24] RAKESH S, TAIBANGNGANBI C N, KUMAR S S, ROY S S, ANSARI M A, PRAKASH N. Genetic diversity of Chilli veinal mottle virus infecting different chilli landraces in North East India indicates the possibility of transboundary movement of virus. 3 Biotech, 2018,8(8): Article number 357.
pmid: 30105182
[25] MURPHY J F, BLAUTH J R, LIVINGSTONE K D, LACKNEY V K, JAHN M K. Genetic mapping of the pvr1 locus in Capsicum spp. and evidence that distinct Potyvirus resistance loci control responses that differ at the whole plant and cellular levels. Molecular Plant-Microbe Interactions, 1998,11(10):943-951.
doi: 10.1094/MPMI.1998.11.10.943
[26] 商文静, 樊小雪, 肖红娟. 转基因抗病毒线辣椒果实品质分析. 西北农业学报, 2001,10(3):24-26.
SHANG W J, FAN X X, XIAO H J. Analysis of fruit quality in transgenic chilli pepper. Acta Agriculturae Boreali-Occidentalis Sinica, 2001,10(3):24-26. (in Chinese)
[27] FERNANDEZ A, GARCIA J A. The RNA helicase CI from plum pox potyvirus has two regions involved in binding to RNA. FEBS Letter, 1996,388(2/3):206-210.
[28] 王继华, 瞿素萍, 孔宝华, 陆琳, 余朝秀. 百合无症病毒的RT-PCR和IC-RT-PCR检测. 云南农业大学学报, 2004,19(2):148-150.
WANG J H, QU S P, KONG B H, LU L, YU C X. Detection of Lily symptomless virus by RT-PCR and IC-RT-PCR. Journal of Yunnan Agricultural University, 2004,19(2):148-150. (in Chinese)
[29] 于翠, 杨翠云, 张舒亚, 乔艳艳. 南芥菜花叶病毒的几种PCR检测方法的建立和比较研究. 植物病理学报, 2008,38(4):388-393.
YU C, YANG C Y, ZHANG S Y, QIAO Y Y. Development and comparative studies of several PCR for detectingArabis mosaic virus. Acta Phytopathologica Sinica, 2008,38(4):388-393. (in Chinese)
[30] 秦文韬, 王忠跃, 张昊. 环介导恒温扩增技术(LAMP)及其在植物病毒检测中的应用. 中国农学通报, 2013,29(21):170-174.
QIN W T, WANG Z Y, ZHANG H. The progress of loop-mediated isothermal amplification (LAMP) and its application in detection of plant virus. Chinese Agricultural Science Bulletin, 2013,29(21):170-174. (in Chinese)
[31] CHIKH-ALI M, MAOKA T, NATSUAKI K T, NATSUAKI T. The simultaneous differentiation ofPotato virus Y strains including the newly described strain PVYNTN-NW by multiplex PCR assay. Journal of Virological Methods , 2010,165(1):15-20.
pmid: 20025905
[32] GAO F L, JIN J, ZOU W C, LIAO F R, SHEN J G. Geographically driven adaptation of Chilli veinal mottle virus revealed by genetic diversity analysis of the coat protein gene. Archives of Virology, 2016,161(5):1329-1333.
pmid: 26831930
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