Development of a Multiplex RT-PCR for the Simultaneous Detection of Three Viruses in Cherry Plants

Park, Chung Youl; Park, Jeongran; Lee, Geunsik; Yi, Seung-In; Kim, Byeong‐Hoon; Eom, Jung Sik; Lee, Som Gyeol; Kim, Hongsup

doi:10.5423/rpd.2018.24.3.233

Cited by 2 publications

(1 citation statement)

References 17 publications

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“…Furthermore, multiplex RT-PCR (mRT-PCR) allows rapid and low-cost detection owing to its ability to simultaneously detect multiple target pathogens in a single reaction (Asano et al 2015;Yao et al 2014). Therefore, diagnostic methods based on mRT-PCR have been developed for the diagnosis of viral pathogens in various plants, such as beans (Park et al 2018a), cherries (Park et al 2018b), garlic (Nam et al 2015), and pears (Kim et al 2019).…”

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confidence: 99%

A survey of viruses and viroids in astringent persimmon (Diospyros kakiThunb.) and the development of a one-step multiplex reverse transcription-polymerase chain reaction assay for the identification of pathogens

et al. 2022

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Astringent persimmon (Diospyros kaki Thunb.) is an important fruit crop in Korea; it possesses significant medicinal potential. However, knowledge regarding the pathogens affecting this crop, particularly, viruses and viroids, is limited. In the present study, reverse transcriptionpolymerase chain reaction (RT-PCR) and high-throughput transcriptome sequencing (HTS) were used to investigate the viruses and viroids infecting astringent persimmons cultivated in Korea. A one-step multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of the pathogens was developed by designing species-specific primers and selecting the primer pairs via combination and detection limit testing. Seven of the sixteen cultivars tested were found to be infection-free. The RT-PCR and HTS analyses identified two viruses and one viroid in the infected samples (n = 51/100 samples collected from 16 cultivars). The incidence of single infections (n = 39/51) was higher than that of mixed infections (n = 12/51); the infection rate of the Persimmon cryptic virus was the highest (n = 31/39). Comparison of the monoplex and mRT-PCR results using randomly selected samples confirmed the efficiency of mRT-PCR for the identification of pathogens. Collectively, the present study provides useful resources for developing disease-free seedlings; further, the developed mRT-PCR method can be extended to investigate pathogens in other woody plants.

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Section: Introductionmentioning

confidence: 99%

A survey of viruses and viroids in astringent persimmon (Diospyros kakiThunb.) and the development of a one-step multiplex reverse transcription-polymerase chain reaction assay for the identification of pathogens

et al. 2022

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First Report of Beet Western Yellows Virus in Radish in Korea

et al. 2023

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Radish (Raphanus sativus L.) is an important root vegetable widely consumed in kimchi in Korea. In October 2021, radish leaves with virus-like symptoms of mosaic and yellowing were collected in three fields around Naju, Korea (Fig. S1). A pooled sample (n = 24) was screened for causal viruses by high-throughput sequencing (HTS), with detection confirmed by reverse transcription (RT) PCR. Total RNA was extracted from symptomatic leaves using the Plant RNA Prep kit (Biocube System, Korea), and a cDNA library was constructed and sequenced on an Illumina NovaSeq 6000 system (Macrogen, Korea). De novo transcriptome assembly yielded 63,708 contigs, which were analyzed against the viral reference genome database in GenBank by BLASTn and BLASTx searches. Two large contigs were clearly of viral origin. BLASTn analysis showed that a 9,842-bp contig (4,481,600 mapped reads, mean read coverage 68,758.6×) had 99% identity (99% coverage) with isolate CCLB of turnip mosaic virus (TuMV) from radish in China (KR153038). A second contig of 5,711 bp (7,185 mapped reads, mean read coverage 189.9×) had 97% identity (99% coverage) with isolate SDJN16 of beet western yellows virus (BWYV) from Capsicum annuum in China (MK307779). To confirm the presence of these viruses, total RNA extracted from 24 leaf samples was subjected to RT-PCR using primers specific for TuMV (N60_5’-ACATTGAAAAGCGTAACCA-3’ and C30_5’-TCCCATAAGCGAGAATACTAACGA-3’, amplicon 356 bp) and BWYV (95F_5’-CGAATCTTGAACACAGCAGAG-3’ and 784R_5’-TGTGGG ATCTTGAAGGATAGG-3’, amplicon 690 bp) for virus detection. Of the 24 samples, 22 were positive for TuMV and 7 were co-infected with BWYV. Single infection of BWYV was not detected. Infection with TuMV, the predominant virus in radish in Korea, was previously reported (Choi and Choi, 1992; Chung et al., 2015). To determine the complete genomic sequence of the BWYV isolate (BWYV-NJ22) from radish, RT-PCR was conducted using eight overlapping primer pairs designed according to the alignment of previously reported BWYV sequences (Table S2). Terminal sequences of the viral genome were analyzed by 5′ and 3′ rapid amplification of cDNA ends (RACE; Thermo Fisher Scientific Corp.). The assembled complete genome sequence of BWYV-NJ22 was 5,694 nt long and was deposited in GenBank (accession no. OQ625515). The Sanger sequences shared 96% nt identity with the HTS-derived sequence. BLASTn analysis showed that BWYV-NJ22 had high nucleotide identity (98%) at the complete genome level with a BWYV isolate (OL449448) from C. annuum in Korea. BWYV (genus Polerovirus, family Solemoviridae), is an aphid-borne virus with a host range that includes > 150 plant species and is one of the most important viruses causing yellowing and stunting of vegetable crops (Brunt et al., 1996; Duffus 1973). In Korea, BWYV was first reported to infect paprika, followed by pepper, motherwort, and figwort (Jeon et al., 2021; Kwon et al., 2016; 2018; Park et al., 2018). During fall and winter 2021, 675 radish plants with virus-like symptoms of mosaic, yellowing, and chlorosis were collected from 129 farms in major cultivation areas in Korea and analyzed by RT-PCR using the BWYV detection primers. The incidence of BWYV in radish plants was 4.7%, and all infections were mixed infections with TuMV. To our knowledge, this is the first report of BWYV infecting radish in Korea. The symptoms of single BWYV infection are unclear, as radish is a new host plant of BWYV in Korea. Further research on the pathogenicity and impact of this virus in radish is therefore needed.

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Development of a Multiplex RT-PCR for the Simultaneous Detection of Three Viruses in Cherry Plants

Cited by 2 publications

References 17 publications

A survey of viruses and viroids in astringent persimmon (Diospyros kakiThunb.) and the development of a one-step multiplex reverse transcription-polymerase chain reaction assay for the identification of pathogens

A survey of viruses and viroids in astringent persimmon (Diospyros kakiThunb.) and the development of a one-step multiplex reverse transcription-polymerase chain reaction assay for the identification of pathogens

First Report of Beet Western Yellows Virus in Radish in Korea

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