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KR-20260065089-A - Marker composition for detecting seedlings virus and uses thereof

KR20260065089AKR 20260065089 AKR20260065089 AKR 20260065089AKR-20260065089-A

Abstract

The present invention relates to a marker composition for detecting viruses in seedlings and the use thereof. Since the marker composition of the present invention can specifically detect BlShV (Blueberry shock virus), BCRV (Blackberry chlorotic ringspot virus), CiLV-C (Citrus leprosis virus C), CPsV (Citrus psorosis virus), and PPV (Plum pox virus), it can contribute to the supply of disease-free plants and quality improvement by blocking viruses that may be introduced through imported seedlings in advance during border quarantine and quarantine cultivation quarantine.

Inventors

  • 김국형
  • 정민휴
  • 김나희
  • 오승현
  • 신용길
  • 장선희
  • 민병은

Assignees

  • 서울대학교산학협력단
  • 대한민국(농림축산식품부 농림축산검역본부장)

Dates

Publication Date
20260508
Application Date
20241031

Claims (7)

  1. Oligonucleotide primer set of SEQ ID NOs 1 and 2 and oligonucleotide probe of SEQ ID NO. 3; oligonucleotide primer set of SEQ ID NOs 1 and 2 and oligonucleotide probe of SEQ ID NO. 4; oligonucleotide primer set of SEQ ID NOs 5 and 6 and oligonucleotide probe of SEQ ID NO. 7; oligonucleotide primer set of SEQ ID NOs 5 and 6 and oligonucleotide probe of SEQ ID NO. 8; oligonucleotide primer set of SEQ ID NOs 9 and 10 and oligonucleotide probe of SEQ ID NO. 11; oligonucleotide primer set of SEQ ID NOs 12 and 13 and oligonucleotide probe of SEQ ID NO. 14; oligonucleotide primer set of SEQ ID NOs 15 and 16 and oligonucleotide probe of SEQ ID NO. 17; oligonucleotide primer set of SEQ ID NOs 18 and 19 and oligonucleotide probe of SEQ ID NO. 20; oligonucleotide primer set of SEQ ID NOs 21 and 22 and oligonucleotide probe of SEQ ID NO. 23; A marker composition for detecting seedling viruses, comprising any one oligonucleotide primer set and probe selected from the group consisting of the oligonucleotide primer set of SEQ ID NOs 24 and 25 and the oligonucleotide probe of SEQ ID NO. 26.
  2. A marker composition for detecting seedling viruses according to claim 1, characterized in that the seedling virus is one or more selected from the group consisting of BCRV (Blackberry chlorotic ringspot virus), BlShV (Blueberry shock virus), CPsV (Citrus psorosis virus), CiLV-C (Citrus leprosis virus C), and PPV (Plum pox virus).
  3. In claim 1, the oligonucleotide primer set of SEQ ID NOs 1 and 2 and the oligonucleotide probe of SEQ ID NO. 3, or the oligonucleotide primer set of SEQ ID NOs 1 and 2 and the oligonucleotide probe of SEQ ID NO. 4 are for the detection of BCRV (Blackberry chlorotic ringspot virus); the oligonucleotide primer set of SEQ ID NOs 5 and 6 and the oligonucleotide probe of SEQ ID NO. 7, or the oligonucleotide primer set of SEQ ID NOs 5 and 6 and the oligonucleotide probe of SEQ ID NO. 8 are for the detection of BLShV (Blueberry shock virus); the oligonucleotide primer set of SEQ ID NOs 9 and 10 and the oligonucleotide probe of SEQ ID NO. 11, or the oligonucleotide primer set of SEQ ID NOs 12 and 13 and the oligonucleotide probe of SEQ ID NO. 14 are for the detection of CPsV (Citrus psorosis virus); A marker composition for detecting seedling viruses, characterized in that the oligonucleotide primer set of SEQ ID NOs 15 and 16 and the oligonucleotide probe of SEQ ID NO. 17, or the oligonucleotide primer set of SEQ ID NOs 18 and 19 and the oligonucleotide probe of SEQ ID NO. 20 are for detecting CiLV-C (Citrus leprosis virus C); and the oligonucleotide primer set of SEQ ID NOs 21 and 22 and the oligonucleotide probe of SEQ ID NO. 23, or the oligonucleotide primer set of SEQ ID NOs 24 and 25 and the oligonucleotide probe of SEQ ID NO. 26 are for detecting PPV (Plum pox virus).
  4. A kit for detecting seedling viruses comprising the marker composition of claim 1 and a reagent for performing an amplification reaction.
  5. In claim 4, the kit comprises a reagent for performing the amplification reaction, the reagent comprising DNA polymerase, dNTPs, and a buffer.
  6. Step of isolating total RNA from a sample suspected of seedling virus infection; A step of amplifying a target sequence by performing RT-PCR (Reverse Transcription Polymerase Chain Reaction) using the total RNA isolated above as a template and the marker composition according to claim 1; and A method for detecting seedling viruses, comprising the step of detecting the product of the amplification step.
  7. A method according to claim 6, wherein the detection of the amplified product is performed via gel electrophoresis, radiometric measurement, fluorescence measurement, phosphorescence measurement, or DNA chip.

Description

Marker composition for detecting seedling viruses and uses thereof The present invention relates to a marker composition for detecting viruses in seedlings and its use. Since the liberalization of global import and export markets, the country's reliance on agricultural imports has been steadily increasing. Given that the primary objective of plant quarantine is to prevent the influx of alien pests, diseases, and weeds, the volume of domestic plant quarantine operations can be seen as higher than ever before. Imported plants are classified according to their intended use into 'planting and planting'—which are directly sown or planted in domestic soil—and 'non-planting'—which are consumed without being planted. Among these, 'planting and planting' plants pose the highest risk of introduction by alien pests. This is particularly critical for plant viruses, as they can remain dormant within the plant body and become active and spread significantly once the plant is planted and grows. When plants are imported, inspection for pests and diseases must be carried out quickly and accurately. Although there are various methods for classifying and identifying plant viral diseases, testing methods that possess the advantages of rapid, precise, and mass testing are preferred in quarantine. Therefore, diagnostic methods such as the ELISA population method, which possesses these advantages relatively well, and electron microscopy, PCR, and bioassay are appropriately utilized depending on the characteristics of each imported crop. The Animal and Plant Quarantine Agency publishes a list of standard testing methods for each plant and major target pathogen, and conducts inspections by designating specific targets. While testing methods for major target viruses are available, there are instances where testing methods have not been established to detect sudden outbreak viruses or viruses for which host plants have not been established. As a result of recently updating the list of pathogens subject to mandatory arrival inspection for seedlings subject to quarantine cultivation, prohibited and controlled-grade quarantine viruses for which real-time PCR testing methods have not been developed were identified. Among the viruses that can be introduced through fruit tree seedlings, such as BCRV (Blackberry chlorotic ringspot virus), BLShV (Blueberry shock virus), CPsV (Citrus psorosis virus), CiLV-C (Citrus leprosis virus C), and PPV (Plum pox virus), precise testing techniques to detect these viruses have not been established. Therefore, it is necessary to develop precise testing techniques based on the TaqMan probe real-time PCR method to establish a precise testing system capable of preemptively blocking them during border quarantine and quarantine cultivation. Meanwhile, Korean Published Patent No. 2017-0059090 discloses 'a primer set for specifically detecting the quarantine bacterium Curtobacterium placumfaciens pv. placumfaciens and its use,' and Korean Published Patent No. 2019-0050135 discloses 'a primer set for specifically detecting the quarantine plant virus avocado sunbrooch viroid and its use,' but there is no description of the marker composition for detecting seedling viruses BCRV, BlShV, CPsV, CiLV-C, and PPV according to the present invention, nor of its use. Figure 1 is a schematic diagram showing the target locations of the five seedling virus-specific primer set and probe (Table 1) of the present invention. Figure 2 shows the results of analyzing species specificity by performing qPCR on the five types of seedling viruses (BlShV, Blueberry shock virus; BCRV, Blackberry chlorotic ringspot virus; CiLV-C, Citrus leprosis virus C; CPsV, Citrus psorosis virus; PPV, Plum pox virus) and other types of viruses (BLMV, Blueberry leaf mottle virus; TRSV, Tobacco ringspot virus; BYVaV, Blackberry yellow vein associated virus; ACLSV, Apple chlorotic leafspot virus; PNSRV, Prunus necrotic ringspot virus; CiMV, Cytomegalovirus; CTLV, Citrus tatter leaf virus; CTV, Citrus tristeza virus; SDV, Satsuma dwarf virus; HSVd, Hop stunt viroid) using the five types of seedling virus-specific primer set and probes of the present invention (Table 1). Figure 3 shows the results of a sensitivity analysis of the five seedling virus-specific probes of the present invention (Table 1). Nine templates were used with DNA copy numbers diluted to 1/10, ranging from n*10^9 copies (red) to n*10^1 copies (blue). The curve shows that amplification occurred at regular intervals from the left (red) of the highest copy number to the right. To achieve the objective of the present invention, the present invention comprises: an oligonucleotide primer set of SEQ ID NOs. 1 and 2 and an oligonucleotide probe of SEQ ID NO. 3; an oligonucleotide primer set of SEQ ID NOs. 1 and 2 and an oligonucleotide probe of SEQ ID NO. 4; an oligonucleotide primer set of SEQ ID NOs. 5 and 6 and an oligonucleotide probe of SEQ ID NO. 7; an oligonucleotide primer set of SEQ ID NOs