Introduction
American plum line pattern virus (APLPV) belonging to the genus Ilarvirus in the family Bromoviridae has a tripartite genome (RNA1, RNA2, and RNA3) (Herranz et al., 2008; Pallas et al., 2013). RNA1 and RNA2 encode proteins associated with viral replication, and RNA3 encodes a movement protein (MP) and a coat protein (CP) (Herranz et al., 2008; Pallas et al., 2013). APLPV was first reported in North America and has been recently recorded in European and Mediterranean regions (Herranz et al., 2008; Candresse et al., 2017). The virus was detected in Prunus mume, P. persica, and P. serrulate, showing chlorotic lines on the leaves (EPPO, 2006). Some cultivars, however, were asymptomatic (Alayasa et al., 2003). APLPV has been listed as an A1 plant pathogen by the European and Mediterranean Plant Protection Organization (EPPO) (Rwahnih et al., 2004). In addition, APLPV has also been designated as a plant quarantine virus in Korea, and some Prunus spp. (plum, peach, apricot, and almond) imported from foreign countries are examined for the presence of this virus (Animal and Plant Quarantine Agency, www.qia.go.kr).
Various methods are used to confirm plant virus infections, such as polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), DNA chip, and high-throughput sequencing technology (Boonham et al., 2014; Jeong et al., 2014). ELISA has been the primary method to detect the presence of plant viruses in the past when molecular biology techniques were not common. Currently, PCR assays are mainly used in various fields, including diagnostic fields, because they require shorter test duration and have better sensitivity and specificity than ELISA (Lee et al., 2013a; 2013b; Shin et al., 2017). Furthermore, because the PCR amplicons can be further analyzed through sequencing technology, more accurate experimental results can be obtained (Lee et al., 2021). Previous studies have attempted the development of various detection methods, including a PCR assay, to detect APLPV (Scott and Zimmerman, 2001; Alayasa et al., 2003; Sanchez-Navarro et al., 2005). However, despite their usability in virus detection, these methods were not developed for the purpose of plant quarantine inspections, as described by Lee et al. (2021). Therefore, in this study, we developed a one-step RT-PCR assay for the detection of APLPV.
Materials and Methods
Collection of samples
To collect virus-positive samples and target plants, we purchased positive and negative controls for ELISA (Agdia, Elkhart, IN, USA) and healthy seeds and seedlings. The plant virus-positive samples were as follows: APLPV, Apple mosaic virus (ApMV), Asparagus virus 2 (AV2), Blueberry shock virus (BlShV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Tobacco streak virus (TSV). The plant samples were as follows: Prunus mume, P. yedoensis, P. persica, P. armeniaca, P. dulcis, P. tomentosa, P. avium, P. glandulosa, P. salicina, and P. cerasifera.
Design of candidate primers
To design candidate primers for the detection of APLPV, all 33 nucleotide sequences (4 in RNA1, 6 in RNA2, and 23 in RNA3) of APLPV registered in the National Center for Biotechnology Information (NCBI) GenBank were collected and aligned using CLC Main Workbench 6 (QIAGEN, Germantown, MD, USA). Conserved regions (candidate sequences) to be designed as primer sequences were searched through analysis of alignments of all the APLPV nucleotide sequences. The candidate sequences were analyzed to verify the binding specificity for APLPV through NCBI BLASTn analysis (https://blast.ncbi.nlm.nih.gov).
Total RNA extraction and RT-PCR condition
Total RNA was extracted from the samples using the RNeasy® Plant Mini Kit (QIAGEN, Hilden, Germany). One-step RT-PCR assay was performed using the total RNA, random N25 primer (50 pmol), each primer set (10 pmol), and AccuPower® RT-PCR PreMix (Bioneer, Daejeon, Korea). At every step for verification, we used three RT-PCR premixture products manufactured by different companies, but only the results derived from using the AccuPower® RT-PCR PreMix were presented as data. The one-step RT-PCR began with cDNA synthesis at 42℃ for 1 h, followed by RT enzyme inactivation at 95℃ for 15 min. PCR was performed for 32 cycles as follows: 94℃ for 30 sec, 60℃ for 30 sec, and 72℃ for 1 min, followed by an additional extension at 72℃ for 5 min.
The four verification stages for selection of optimal primer sets
In this study, we developed a one-step RT-PCR assay for the detection of APLPV. The expected primer characteristics were as follows: (i) the primers used should be able to accurately detect almost all existing isolates with genetically divergent sequences; (ii) the primers should not bind nonspecifically with the genome of other closely related viruses or (iii) imported plants to be tested; and (iv) finally, the selected primers should have a high detection ability even at a low titer of the target virus.
Development of a positive clone
The sequences of each pair of primers were arranged according to their expected amplicon sizes. The positive clone comprised sequences of primers for detection of six plant viruses including APLPV and artificial sequences unrelated to plant viruses. An artificial DNA fragment was synthesized on the basis of DNA synthesis technology and cloned into the pUCIDT-AMP vector by Integrated DNA Technologies, Inc. (Coralville, IA, USA).
Results and Discussion
Pairwise comparisons between APLPV isolates and combinations of candidate primers
Viruses have sequence variation among isolates within each species (Lim et al., 2015; 2019; Oh et al., 2019), so this point should be taken into account when developing diagnostic methods. Prior to primer design, pairwise comparisons of the sequences of MP and CP genes of RNA3 were performed to analyze the genetic variation across APLPV isolates. The pairwise comparisons showed > 97% nucleotide sequence homologies in the MP and CP nucleotide sequences (Fig. 1). We searched conserved regions based on alignments of the nucleotide sequences of APLPV isolates, and seven conserved regions in RNA2 and eight in RNA3 were searched for primer design. Since RNA1 has a relatively small number of registered nucleotide sequences, its regions were not searched. The BLASTn searches of the 15 candidate sequences revealed the binding specificity of candidate sequence no. 6 for Ageratum latent virus (another member of the genus Ilarvirus) as well as APLPV (Supplementary Table 1). The candidate sequences were matched to generate amplicons of expected size (300 - 900 bp), but candidate sequence no. 7 was not available. Consequently, a total of 15 candidate primer sets were developed (Supplementary Table 2 and Supplementary Fig. 1).
Fig. 1. Pairwise comparisons using complete sequences of MP and CP genes of APLPV isolates. (A) A pairwise comparison based on an alignment of 11 complete MP-coding sequences, and (B) a pairwise comparison based on an alignment of 11 complete CP-coding sequences. The blue and red colors represent the comparison gradient, with relatively low homology in blue and high homology in red. MP, movement protein; CP, coat protein; APLPV, American plum line pattern virus.
The first verification stage
First, the specificity of the candidate primer sets was verified using positive and negative controls for APLPV. In this verification stage, 13 candidate primer sets (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15) were selected (Supplementary Fig. 2). Primer sets 1 and 2, which produced a weak target band and nonspecific reaction in the other RT-PCR premixture tests (data not shown), were excluded.
The second verification stage
Afterward, we confirmed whether the candidate primer sets could nonspecifically bind to the genomes of other closely related viruses. In this verification stage, six ilarviruses (ApMV, AV2, BlShV, PDV, PNRSV, and TSV) were used. The results of the one-step RT-PCR revealed that the use of five candidate primer sets (3, 4, 5, 6, and 12) resulted in nonspecific smear bands (Fig. 2). Consequently, eight candidate primer sets (7, 8, 9, 10, 11, 13, 14, and 15) were selected for the subsequent stages of verification.
Fig. 2. Polymerase chain reaction results for nonspecific reactions in six ilarviruses. Six closely related viruses belonging to the genus Ilarvirus (Apple mosaic virus, Asparagus virus 2, Blueberry shock virus, Prune dwarf virus, Prunus necrotic ringspot virus, and Tobacco streak virus) were examined to confirm the occurrence of nonspecific reactions.
The third verification stage
Some Prunus species (P. mume, P. yedoensis, P. persica, P. armeniaca, P. dulcis, P. tomentosa, P. avium, P. glandulosa, P. salicina, and P. cerasifera) imported into Korea are tested for APLPV infections. During plant quarantine inspections, nonspecific products derived from the host plants may interfere with the test results. Therefore, we confirmed whether nonspecific amplicons were generated in the RT-PCRs using the target plant samples. Primer sets 8 and 13 were excluded because of nonspecific band formation (Fig. 3). In addition, four primer sets (10, 11, 14, and 15) were excluded because of observed inconsistent reactivity such as weak reactivity from the APLPV-infected sample (Fig. 3). Thus, two candidate primer sets (7 and 9) were selected.
The fourth verification stage
Finally, concentration-dependent amplification tests were performed using primer sets 7 and 9. The initial amount of total RNA extracted from the APLPV-infected sample was 100 ng, and six additional templates were prepared through 1/10 serial dilutions. Primer sets 7 and 9 generated amplicons up to 1 and 0.1 ng of the total RNA template, respectively (Supplementary Fig. 3). In this study, we used the positive samples for ELISA because of the difficulty associated with collecting and handling plants infected with APLPV, a virus designated and regulated as a plant quarantine pathogen in Korea. Considering that the positive control was a freeze-dried sample, sensitivity in fresh samples is expected to be much higher.
Fig. 3. Polymerase chain reaction results for nonspecific reactions in 10 target plants. In total, 10 Prunus spp. (P. mume, P. yedoensis, P. persica, P. armeniaca, P. dulcis, P. tomentosa, P. avium, P. glandulosa, P. salicina, and P. cerasifera), which are target imported plants for American plum line pattern virus (APLPV) inspections in Korean plant quarantine, were examined to confirm the occurrence of nonspecific reactions.
Verification for efficiency of positive clone
The use of positive and negative controls in PCR assays can improve the reliability of the inspection process and results. However, it is challenging to obtain plant quarantine pathogens through collection and purchase. Moreover, laboratory contamination due to the frequent use of nucleic acids extracted from positive samples makes it difficult to analyze the test results. To solve this problem, we developed a plasmid using DNA synthesis technology for use as a positive control in PCR (Fig. 4a). To verify the use of the plasmid as a positive control in PCR, PCR was performed with 1 and 0.1 ng of the positive clone. The two primer sets (no. 7 and 9) generated PCR products of the expected sizes without any nonspecific bands (Fig. 4b). Thus, the positive clone could be used for the two APLPV-specific primer sets and could additionally be applied to primer sets for detecting five other viruses (data not shown). This result indicated that the positive clone developed on the basis of DNA synthesis technology can be used efficiently as a positive PCR control in PCR assay including plant quarantine inspections.
Fig. 4. Development of a positive clone for use as a positive control in PCR for APLPV detection. (A) The positive clone was synthesized by arranging primer sequences that correspond with the expected PCR amplicon sizes. (B) Concentration-dependent amplification was performed using 1 and 0.1 ng of the positive clone. PCR, polymerase chain reaction; APLPV, American plum line pattern virus.
Conclusion
In this study, we selected two primer sets to detect APLPV and developed a positive clone for use as the control template in an RT-PCR assay. The two primer sets and the positive clone can be efficiently used to detect the APLPV infection of target imported plants in Korean plant quarantine inspections. Furthermore, the primer sets designed and examined in this study will be useful for APLPV detection in various other fields.
Ethical approval
This article does not describe any studies with human participants or animals performed by any of the authors.
Acknowledgments
This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET), funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (320044-03-1-SB010). We thank Edanz for editing the English text of a draft of this manuscript.
Supplementary Data
Supplementary Fig. 1. Development of a positive clone for use as a positive control in PCR for APLPV detection. (A) The positive clone was synthesized by arranging primer sequences that correspond with the expected PCR amplicon sizes. (B) Concentration-dependent amplification was performed using 1 and 0.1 ng of the positive clone. PCR, polymerase chain reaction; APLPV, American plum line pattern virus.
