百合中车前草花叶病毒的实时荧光定量PCR检测

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

摘要/Abstract

摘要：

车前草花叶病毒（Plantago asiatica mosaic virus,PlAMV）病是侵染百合等作物的危险性病害,亟待建立其监测防控体系,以防止其扩散传播。根据PlAMV的外壳蛋白（Coat protein,CP）基因的保守序列设计特异性检测引物,通过对引物浓度和反应退火温度进行优化,建立了以CP-2 F/R为特异引物,引物浓度为10 μmol · L^-1,退火温度为59.4 ℃的高效实时荧光定量PCR检测技术。该检测技术特异性强,灵敏度是RT-PCR的100倍,病毒最低检出浓度为13 copies · μL^-1。对75份田间百合样品进行检测,结果显示：RT-qPCR方法检测（29份,38.67%）高于RT-PCR检测出的阳性样品（21份,28.00%）。

关键词: 百合, 车前草花叶病毒, 实时荧光定量PCR

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

中图分类号:

S682.2

宋蒙, 徐雷锋, 曹雨薇, 杨盼盼, 毕蒙蒙, 何国仁, 唐玉超, 王静, 明军. 百合中车前草花叶病毒的实时荧光定量PCR检测[J]. 园艺学报, 2021, 48(12): 2497-2505.

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.

图/表 11

图1 感染车前草花叶病毒的百合

Fig. 1 Lily infected with PlAMV

表1 检测PlAMV实时荧光定量PCR引物序列

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

图2 PlAMV引物CP-2 F/R在4个感染PlAMV百合样品（1 ~ 4）中的RT-PCR扩增结果

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

图3 不同引物浓度下PlAMV的RT-qPCR扩增

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

图4 不同退火温度条件下PlAMV溶解曲线

Fig. 4 Melting curves of PlAMV at different annealing temperature

图5 不同退火温度下PlAMV的RT-qPCR扩增

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

图6 PlAMV的RT-qPCR标准曲线

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

图7 不同浓度PlAMV质粒的RT-PCR检测结果 M：Marker;Lane 2 ~ 9：10倍比稀释的模板;N：阴性对照。

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.

图8 不同浓度PlAMV质粒的RT-qPCR扩增曲线

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

图9 RT-qPCR特异性检测扩增曲线（A）和熔解峰图（B）

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

图10 卷丹不同部位PlAMV RT-qPCR检测

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

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