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;;


【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"

Upper and lower primer sequences (5′-3′)
Length (nt)
Tm (℃)
Expected size (bp)


Table 2

Different gradient combinations and the primer dosage"

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"

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