香石竹斑驳病毒属简并引物RT-PCR检测方法的建立

doi:10.3864/j.issn.0578-1752.2023.12.005

Abstract

Abstract:

【Objective】The objective of this study is to establish a rapid, sensitive, and broad-spectrum screening method for simultaneous detection and identification of the genera Alphacarmovirus, Betacarmovirus, and Gammacarmovirus using degenerate primer RT-PCR combined with sequence analysis.【Method】Multiplexed analysis of genome sequences was aligned to search for suitable conserved regions for the design of the degenerate primers. One pair of degenerate primer Carmo-F2/Carmo-R2 was designed based on the RNA-dependent RNA polymerase (RdRp) gene sequences, and another pair primer Carmo-F2a/Carmo-R2a was formed by adding the non-complementary AT-rich sequences (AATAAATCATAA) to the 5′ end of the degenerate primers. The broad-spectrum, specificity, and sensitivity of RT-PCR method were analyzed. The sequencing, BLASTn analysis and phylogenetic analysis of PCR products were performed. The method was used to screen and detect viruses on Chinese hibiscus (Hibiscus rosa-sinensis) samples from Xiamen, China.【Result】Degenerate primers Carmo-F2/Carmo-R2 and Carmo-F2a/Carmo-R2a were used to amplify partial RdRp gene of the members of genera Alphacarmovirus, Betacarmovirus, and Gammacarmovirus by RT-PCR. The fragment of approximately 500 and 550 bp was amplified, respectively. The developed RT-PCR assay was successfully used to detect angelonia flower break virus (AnFBV; Alphacarmovirus), calibrachoa mottle virus (CbMV; Alphacarmovirus), carnation mottle virus (CarMV; Alphacarmovirus), and pelargonium flower break virus (PFBV; Alphacarmovirus), hibiscus chlorotic ringspot virus (HCRSV; Betacarmovirus), melon necrotic spot virus (MNSV; Gammacarmovirus). The specificity test showed that no specific band could be obtained from maize chlorotic mottle virus (MCMV; Machlomovirus), pelargonium line pattern virus (PLPV; Pelarspovirus), carnation ringspot virus (CRSV; Dianthovirus), and healthy plants which including watermelon, melon, pumpkin, soybean, and pea. The sensitivity results showed that the primers Carmo-F2/Carmo-R2 could be detected up to 10^-2 dilution and the primers Carmo-F2a/Carmo-R2a could be detected up to 10^-3 dilution, which indicated that the non-complementary AT-rich sequences added at the 5′ end of the degenerate primers could increase the sensitivity of degenerate primer RT-PCR. BLAST analysis showed that the sequences determined had the highest sequence consistency with the corresponding virus species. Phylogenetic analysis based on partial amino acid sequences of the RdRp gene showed that it is consistent with the current classification of the subfamily Procedovirinae, and the viruses could be identified at the species level. HCRSV was detected in all 13 H. rosa-sinensis samples with suspected virus symptoms.【Conclusion】The RT-PCR method based on the degenerate primers Carmo-F2/Carmo-R2 and Carmo-F2a/Carmo-R2a can be used for the screening and detection of viruses of the genera Alphacarmovirus, Betacarmovirus, and Gammacarmovirus, and can be used for rapid identification of virus species in combination with sequence analysis and phylogenetic analysis, and may be used to discover the new virus. H. rosa-sinensis plants were infested with HCRSV in Xiamen, China.

Key words: Alphacarmovirus, Betacarmovirus, Gammacarmovirus, degenerate primer, RT-PCR, Hibiscus rosa-sinensis, hibiscus chlorotic ringspot virus (HCRSV)

LIAO FuRong, CHEN HongYun, SHEN JianGuo, FANG ZhiPeng, HUANG PengYing, CHEN Qing, LIN LingLing, HONG Jun. Development of a Degenerate Primer RT-PCR Assay for Detection of Carmovirus[J].Scientia Agricultura Sinica, 2023, 56(12): 2288-2301.

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病毒Virus	分离物Isolate	提供者Provider
甲型香石竹斑驳病毒属Alphacarmovirus
香彩雀花碎色病毒Angelonia flower break virus (AnFBV)	LPC36600	美国Agdia, USA
小花矮牵牛斑驳病毒Calibrachoa mottle virus (CbMV)	LPC83000	美国Agdia, USA
CbMV	07008PC	德国Loewe, Germany
香石竹斑驳病毒Carnation mottle virus (CarMV)	LPC68000	美国Agdia, USA
CarMV	07057PC	德国Loewe, Germany
天竺葵花碎色病毒Pelargonium flower break virus (PFBV)	LPC90000	美国Agdia, USA
PFBV	07073PC	德国Loewe, Germany
乙型香石竹斑驳病毒属Betacarmovirus
木槿褪绿环斑病毒Hibiscus chlorotic ringspot virus (HCRSV)	LPC12300	美国Agdia, USA
丙型香石竹斑驳病毒属Gammacarmovirus
甜瓜坏死斑点病毒Melon necrotic spot virus (MNSV)	LPC12402	美国Agdia, USA
MNSV	07097PC	德国Loewe, Germany
玉米褪绿斑驳病毒属Machlomovirus
玉米褪绿斑驳病毒Maize chlorotic mottle virus (MCMV)	LPC17002	美国Agdia, USA
天竺葵环斑病毒属Pelarspovirus
天竺葵线纹病毒Pelargonium line pattern virus (PLPV)	07094PC	德国Loewe, Germany
石竹属病毒属Dianthovirus
香石竹环斑病毒Carnation ringspot virus (CRSV)	P20-27	中国CAIQ, China*

病毒Virus	登录号GenBank accession number
甲型香石竹斑驳病毒属Alphacarmovirus
侧金盏花属花叶病毒Adonis mosaic virus	LC171345
香彩雀花碎色病毒Angelonia flower break virus	NC_007733
小花矮牵牛斑驳病毒Calibrachoa mottle virus	NC_021926
香石竹斑驳病毒Carnation mottle virus	NC_001265, AF192772, AJ811998, FJ843021, HQ660513, KR002041
金银花环斑病毒Honeysuckle ringspot virus	NC_014967, HQ677625
羽扇豆脉明病毒Nootka lupine vein-clearing virus	NC_009017
天竺葵花碎色病毒Pelargonium flower break virus	NC_005286, DQ256073
巨人柱仙人掌病毒Saguaro cactus virus	NC_001780
乙型香石竹斑驳病毒属Betacarmovirus
碎米荠褪绿斑驳病毒Cardamine chlorotic fleck virus	NC_001600
木槿褪绿环斑病毒Hibiscus chlorotic ringspot virus	NC_003608
日本鸢尾坏死环斑病毒Japanese iris necrotic ring virus	NC_002187
芜菁皱缩病毒Turnip crinkle virus	NC_003821
丙型香石竹斑驳病毒属Gammacarmovirus
豇豆斑驳病毒Cowpea mottle virus	NC_003535
甜瓜坏死斑点病毒Melon necrotic spot virus	NC_001504, JX879088
豌豆茎坏死病毒Pea stem necrosis virus	NC_004995
大豆黄斑驳花叶病毒Soybean yellow mottle mosaic virus	NC_011643

名称Name	序列Sequence (5′-3′) *	位置Position in AF192772
Carmo-F2	GAYCCDGCHSCNMGDRTNAT	1276-1295
Carmo-R2	ACRCARTCRTCNCCRTTRTTNA	1754-1775
Carmo-F2a	AATAAATCATAAGAYCCDGCHSCNMGDRTNAT	1276-1295
Carmo-R2a	AATAAATCATAAACRCARTCRTCNCCRTTRTTNA	1754-1775

名称Name	序列Sequence (5′-3′) *	位置Position in AF192772	说明Note
Carmo-II	AARGTCGACCCGWNCCNMGNGTNATHCAACC	1276-1301	原序列Original sequence^[29]
Carmo-VI	GMMCTGCAGNACRCARTCRTCNCCRTTRTT	1756-1776	原序列Original sequence^[29]
Carmo-II2	AARGTCGAYCCDGCHSCNMGDRTNATHMRDCC	1276-1301	修订后序列Modified sequence
Carmo-VI2	GMMCTGCAVNACRCARTCRTCNCCRTTRTT	1756-1776	修订后序列Modified sequence

病毒种类，分离物Virus species, isolate	长度Length (bp)	序列一致性Nucleotide sequence identity
甲型香石竹斑驳病毒属Alphacarmovirus
AnFBV, LPC36600 (Agdia)	461	94.36% to NC_007733.2, AnFBV
CbMV, LPC83000 (Agdia)	458	99.13% to OK181769.1, CbMV
CbMV, 07008PC (Loewe)	458	99.78% to OK181769.1, CbMV
CarMV, LPC68000 (Agdia)	/	/
CarMV, 07057PC (Loewe)	458	99.56% to MT682299.1, CarMV
PFBV, LPC90000 (Agdia)	461	97.61% to OP357950.1, PFBV
PFBV, 07073PC (Loewe)	461	97.83% to DQ256073.1, PFBV
乙型香石竹斑驳病毒属Betacarmovirus
HCRSV, LPC12300 (Agdia)	458	99.55% to MT512573.1, HCRSV
丙型香石竹斑驳病毒属Gammacarmovirus
MNSV, 07097PC (Loewe)	464	98.71% to OK558728.1, MNSV

Development of a Degenerate Primer RT-PCR Assay for Detection of Carmovirus

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分离物 Isolate	与HCRSV的序列一致性 Nucleotide sequence identity with HCRSV
XL3	90.39% (MN080500)-98.47% (MK279671)
XM1	88.86% (MN080500)-96.72% (KC876666)
XM2	91.48% (DQ392986)-97.60% (OP779319)
XM3	89.96% (MN080500)-96.29% (MK279671)
XM4	89.96% (MN080500)-96.51% (MK279671)
XM5	89.52% (MN080500)-96.29% (MK279671)
XM6	88.86% (MN080500)-96.28% (KC876666)
XM7	90.17% (MN080500)-99.34% (KY933060)
XM8	89.96% (MN080500)-98.69% (KY933060)
XM9	90.13% (MN080500)-96.05% (MK279671)
XM11	90.13% (MN080500)-96.05% (OK636421)
XM12	90.39% (MN080500)-97.60% (MK279671)
XM13	90.39% (MN080500)-97.82% (MK279671)

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