福建蝴蝶兰病毒小RNA测序及RT-PCR检测

doi:10.16420/j.issn.0513-353x.2024-0192

摘要/Abstract

摘要：

从福建省福州、厦门、漳州、泉州采集56个蝴蝶兰（Phalaenopsis）疑似病毒样品，按侵染症状分为褪绿、黄化褐斑、黄化皱缩等3类样品，采用小RNA测序技术和RT-PCR进行检测，共发现8种病毒，按照检出率从高到低分别为建兰花叶病毒（Cymbidium mosaic virus，CyMV）85.71%，齿兰环斑病毒（Odontoglossum ringspot virus，ORSV）35.71%，白三叶草花叶病毒（white clover mosaic virus，WCMV）32.14%，淮山药X病毒（yam virus X，YaVX）21.43%，水仙花叶病毒（Narcissus mosaic virus，NaMV）10.71%，凤果花叶病毒（pepino mosaic virus，PeMV）8.93%，马铃薯X病毒（potato virus X，PoVX）7.14%和仙人指X病毒（Schlumbergera virus X，ScVX）7.14%。其中，CyMV、ORSV和WCMV的检出率在30%以上，多为复合侵染，侵染率达75.51%。以小RNA测序序列为模板，设计出特异引物，建立了同时检测5种病毒的多重RT-PCR体系；ORSV、CyMV、WCMV、YaVX和PoVX的引物对浓度分别为0.30，0.06，0.20，0.50和0.40 μmol · L^-1，退火温度 56 ℃时，可同时扩增出片段大小分别为1 156、908、561、292和162 bp 的目的条带，特异性良好。灵敏度检测结果显示，可从≥0.0001 mg的感病植物组织中检测到这5种病毒。

关键词: 蝴蝶兰, 小RNA深度测序, 多重RT-PCR, 病毒

Abstract:

This study collected 56 suspected virus samples of Phalaenopsis from Fuzhou，Xiamen，Zhangzhou，and Quanzhou in Fujian Province，which were divided into three types of mixed samplesbased on infection symptoms：chlorosis，yellowing brown spots，and yellowing wrinkles. Small RNA sequencing technology and RT-PCR were used for detection，and a total of 8 viruses were found. According to the detection rate，they were Cymbidium mosaic virus（CyMV）（85.71%），Odontoglossum ringspot virus （ORSV）（35.71%），white clover mosaic virus（WCMV）（32.14%），yam virus X（YaVX）（21.43%），Narcissus mosaic virus（NaMV）（10.71%），pepino mosaic virus（PeMV）（8.93%），potato virus X（PoVX）（7.14%），and Schlumbergera virus X（ScVX）（7.14%）from high to low. Among them， detection rates of CyMV，ORSV and WCMV are over 30%，and most of them are compound infections，accounting for 75.51% of the total infections. Using small RNA sequencing sequences as templates，specific primers were designed and a multiplex RT-PCR system was established for simultaneous detection of five viruses. The primer concentrations for ORSV，CyMV，WCMV，YaVX，and PoVX were 0.30，0.06，0.20，0.50 and 0.40 μmol · L^-1，respectively. At an annealing temperature of 56 ℃，target bands with fragment sizes of 1 156，908，561，292 and 162 bp，respectively，can be simultaneously amplified with good specificity. The sensitivity test results show that these 5 viruses can be detected from infected plant tissues equivalent to or greater than 0.0001 mg.

Key words: Phalaenopsis, small RNA deep sequencing, multiple RT-PCR, virus

樊荣辉, 吴建设, 冯子楠, 钟声远, 钟淮钦. 福建蝴蝶兰病毒小RNA测序及RT-PCR检测[J]. 园艺学报, 2025, 52(2): 503-512.

FAN Ronghui, WU Jianshe, FENG Zinan, ZHONG Shengyuan, ZHONG Huaiqin. Detection and Identification of Virus Infecting Phalaenopsis Based on Small RNA Deep Sequencing Technology and Multiple RT-PCR Identification[J]. Acta Horticulturae Sinica, 2025, 52(2): 503-512.

图/表 13

图1 蝴蝶兰病毒病叶片症状

Fig. 1 Symptoms of Phalaenopsis virus disease on leaves

表1 用于检测 8种病毒的引物

Table 1 Primers used to detect 8 viruses

引物 Primer	引物序列（5ʹ-3ʹ）Primer sequence	目的片段/bp Product
CyMV	F：AGTCTCAACCTGACGTGGAG；R：GGGGAAGCGGTGAATATTA	908
YaVX	F：GGCAGAATCTATTGGAAAAA；R：CCATGAATGACTAAGGGAAG	292
NaMV	F：TGTCACTCACATTCCCCCTG；R：TCCTGAACGTTTGCCTCAAC	234
ORSV	F：TATGACAAATCCCAGAACGA；R：GCAAATAGAAACAAACTCCA	1 156
PeMV	F：ATCTTAGCAACCAGCGTTAT；R：GTCCCAAGCAACATAGGC	397
PoVX	F：GAGGGTCCCACTTTTGATGC；R：CAAGCGGTTGGTGATTTTAT	162
ScVX	F：CAATCACCGGACTTTGACCC；R：AATAGCTCCCAGTTTTTCCG	84
WCMV	F：GTTGTGCTCCCTGTTGAA；R：GTCCCAAATGGATAAGGATAG	561

表2 多重RT-PCR引物不同浓度组合

Table 2 Combination of primer concentration of the multiplex RT-PCR μmol · L-1

组合Composition	CyMV	WCMV	YaVX	ORSV	PoVX
1	0.10	0.10	0.10	0.10	0.10
2	0.02	0.40	0.40	0.20	0.20
3	0.02	0.20	0.30	0.10	0.10
4	0.10	0.40	0.40	0.20	0.40
5	0.06	0.20	0.50	0.30	0.40

表3 蝴蝶兰病叶样本小RNA测序信息

Table 3 Small RNA sequencing information in diseased leaf samples of Phalaenopsis

病毒Virus	数量 Contigs count
病毒Virus	褪绿 Chlorosis	黄化褐斑 Yellowing brown spots	黄化皱缩 Yellowing wrinkles
建兰花叶病毒Cymbidium mosaic virus（CyMV）	44	13	25
水仙花叶病毒Narcissus mosaic virus（NaMV）	0	1	0
齿兰环斑病毒Odontoglossum ringspot virus（ORSV）	0	12	0
凤果花叶病毒pepino mosaic virus（PeMV）	0	1	0
马铃薯X病毒potato virus（PoVX）	0	1	0
仙人指X病毒Schlumbergera virus X（ScVX）	0	1	0
白三叶草花叶病毒white clover mosaic virus（WCMV）	0	3	2
淮山药X病毒yam virus X（YaVX）	0	3	1

图2 蝴蝶兰病叶样本Clean reads序列长度分布

Fig. 2 Length distribution of Clean reads sequence in diseased leaf samples of Phalaenopsis

表4 蝴蝶兰病叶样本小RNA测拼接序列注释统计结果

Table 4 Annotate statistical results of Small RNA sequencing and splicing sequence in diseased leaf samples of Phalaenopsis

样本Sample	原始数据Raw_reads	待分析数据Clean_reads	GC/%	Q30/%	重叠群N50 Contigs_N50	重叠群数量Contigs_Num
褪绿Chlorosis	13 558 557	10 586 409	50.25	97.20	74	82
黄化褐斑Yellowing brown spots	19 031 729	15 892 227	49.80	97.77	795	37
黄化皱缩 Yellowing wrinkles	13 710 865	10 084 402	50.63	97.74	455	44

图3 蝴蝶兰病叶样品中病毒的RT-PCR检测

Fig. 3 RT-PCR validation of virus in diseased leaf samples of Phalaenopsis 1：CyMV；2：PoVX；3：ORSV；4：YaVX；5：NaMV；6：PeMV；7：ScVX；8：WCMV

表5 蝴蝶兰56份病样的RT-PCR检测结果

Table 5 The viruses in 56 samples detected by RT-PCR

病毒Virus	检出率/% Detection rate
病毒Virus	福州 Fuzhou	泉州 Quanzhou	厦门 Xiamen	漳州 Zhangzhou	合计Total
CyMV	86.67	84.62	78.57	92.86	85.71
NaMV	13.30	7.70	0	21.43	10.71
ORSV	40.00	23.08	35.71	42.86	35.71
PeMV	13.33	0	7.14	7.14	7.14
PoVX	0	15.38	14.29	7.14	8.93
ScVX	0	7.70	21.43	7.14	8.93
WCMV	40.00	23.08	28.57	35.71	32.14
YaVX	20.00	23.08	28.57	14.29	21.43

图4 福建省蝴蝶兰各产区病毒混合侵染情况

Fig. 4 Mixed infection of viruses in different Phalaenopsis-growing regions of Fujian Province

图5 蝴蝶兰ORSV、CyMV、WCMV、YaVX和PoVX病毒引物浓度组合多重RT-PCR扩增结果 1-5：不同引物浓度组合（表2）

Fig. 5 Multiplex RT-PCR amplification of ORSV，CyMV，WCMV，YaVX and PoVX with different concentration of primer in Phalaenopsis 1-5：Combinations of different primer concentration（Table 2）

图6 蝴蝶兰ORSV、CyMV、WCMV、YaVX和PoVX病毒不同退火温度多重RT-PCR扩增结果

Fig. 6 Multiplex RT-PCR amplification of ORSV，CyMV，WCMV，YaVX and PoVX with different annealing temperatures in Phalaenopsis

图7 蝴蝶兰ORSV、CyMV、WCMV、YaVX和PoVX病毒多重RT-PCR灵敏度分析

Fig. 7 Sensitivity analysis of multiple RT-PCR for ORSV，CyMV，WCMV，YaVX and PoVX in Phalaenopsis

图8 蝴蝶兰ORSV、CyMV、WCMV、YaVX和PoVX病毒的单重及多重RT-PCR

Fig. 8 Detection of ORSV，CyMV，WCMV，YaVX and PoVX in Phalaenopsis by individual and multiplex RT-PCR

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