Real-time Fluorescence Quantitative PCR Detection of Plantago Asiatica Mosaic Virus in Lilies

doi:10.16420/j.issn.0513-353x.2021-0147

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (12): 2497-2505.doi: 10.16420/j.issn.0513-353x.2021-0147

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Real-time Fluorescence Quantitative PCR Detection of Plantago Asiatica Mosaic Virus in Lilies

SONG Meng, XU Leifeng(), CAO Yuwei, YANG Panpan, BI Mengmeng, HE Guoren, TANG Yuchao, WANG Jing, MING Jun()

Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China

Received:2021-04-12 Revised:2021-11-25 Published:2022-01-04
Contact: XU Leifeng,MING Jun E-mail:xuleifeng@caas.cn;mingjun@caas.cn

Abstract

Abstract:

As a potentially harmful virus,plantago asiatica mosaic virus（PlAMV）has been reported to infect lilies and other crops in China,so it is urgent to establish a monitoring and control system to prevent its spread. In this study,specific primer pair CP-2F/R were designed based on the conserved region of coat protein（CP）gene of PlAMV. By optimizing concentration of primers and annealing temperature,an efficient real-time fluorescent quantitative polymerase chain reaction（RT-qPCR）was established with optimal primer concentration of 10 μmol · L^-1 and optimal annealing temperature of 59.4℃. The sensitivity of this assay was 100 times higher than that of conventional RT-PCR,and the limit of detection by RT-qPCR was 13 copies · μL^-1. The established RT-qPCR was used to detect PlAMV in 75 lily samples,with conventional RT-PCR used as a reference method,and the results showed that more positive samples were detected by RT-qPCR （29,38.67%）than by RT-PCR（21,28.00%）.

Key words: lily, plantago asiatica mosaic virus, real-time fluorescence quantitative PCR

CLC Number:

S682.2

SONG Meng, XU Leifeng, CAO Yuwei, YANG Panpan, BI Mengmeng, HE Guoren, TANG Yuchao, WANG Jing, MING Jun. Real-time Fluorescence Quantitative PCR Detection of Plantago Asiatica Mosaic Virus in Lilies[J]. Acta Horticulturae Sinica, 2021, 48(12): 2497-2505.

Figures/Tables 11

Fig. 1 Lily infected with PlAMV

Table 1 Real-time fluorescence quantitative PCR primer sequences of PlAMV were detected

引物名称 Primer name	引物序列（5′-3′） Primer		产物大小/bp Product size
CP-1	F：GACACGATCACACGAGGCTT	R：GCGATTTGGGCCAGAGAGAT	161
CP-2	F：TCTTCGATGGCCTCCTCAAC	R：TAGGGATCGTGCCGTCTCAT	104
CP-3	F：CTGGACACGATCACACGAGG	R：CAGGGTGGTGGTGACTTTCA	192
CP-4	F：GGCTTTCCAGAAATCTCCCA	R：CTCATTGGCAGTTACTTCGT

Fig. 2 RT-PCR amplification results of PlAMV primer CP-2 F/R in four lily samples（1-4）infected with PlAMV

Fig. 3 RT-qPCR amplification of PlAMV with different primers concentration

Fig. 4 Melting curves of PlAMV at different annealing temperature

Fig. 5 RT-qPCR amplification of PlAMV with primers at different temperatures

Fig. 6 The standard curve of RT-qPCR for PlAMV

Fig. 7 Detection of different PlAMV plasmid concentrations by RT- PCR M：Marker;Lane 2-9：Ten-fold serial dilutions of plasmid;N：Negative control.

Fig. 8 RT-qPCR amplification curve of PlAMV with diqfferent plasmid concentrations

Fig. 9 Amplification curve（A）and melting curve（B）specific detection of RT-qPCR

Fig. 10 Detection of PlAMV in different parts of Lilium lancifolium by RT-qPCR

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Real-time Fluorescence Quantitative PCR Detection of Plantago Asiatica Mosaic Virus in Lilies

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