All Issue

2020 Vol.65, Issue 4 Preview Page
Development of Real-time Quantitative PCR Assay based on SYBR Green I and TaqMan Probe for Detection of Apple Viruses

사과 바이러스 검정을 위한 SYBR Green I 및 TaqMan probe 기반의 real-time PCR 검사법 개발

Seong Heo1
,
Yong Suk Chung2*
허 성1
,
정 용석2*
1Assistant Professor, Department of Horticulture, Kongju National University
2Assistant Professor, Department of Plant Resources and Environment, Jeju National University
1공주대학교 산업과학대학 원예학과 교수
2제주대학교 생명자원과학대학 식물자원환경전공 교수
*Corresponding Author
1 December 2020. pp. 496-507
PDF XML Citation
Abstract

Virus infections of apples result in lowered commercial qualities such as low sugar content, weakened tree vigor, and malformed fruits. An effective way to control viruses is to produce virus-free plants based on the development of an accurate and sensitive diagnostic method. In this study, real-time PCR assays based on SYBR Green I and TaqMan probes were developed for detecting ASGV, ASPV, and ApMV viruses. These methods can detect and quantify 103 to 1011 RNA copies/μL of each virus separately. Compared with methods with two different dyes, the SYBR Green I-based method was efficient for virus detection as well as for assay using the TaqMan probe. Field tests demonstrated that real-time PCR methods developed in this study were applicable to high-throughput diagnoses for virus research and plant quarantine.

Keywords
multiplex RT-PCR
real-time PCR
singleplex RT-PCR
SYBR Green I
TaqMan probe
References
1
Chang, S., J. Puryear, and J. Cairney. 1993. A simple and efficient method for isolation RNA from pine trees. Plant Molecular Biology Reporter 11 : 113-116. 10.1007/BF02670468
2
Chae, J. H. 2016. An improvement plan for nursery tree production and distribution. Daegu Gyeongbuk Development Institute, South Korea. pp. 1-22 (In Korean).
3
Hassan, M., A. Myrta, and J. Polak. 2006. Simultaneous detection and identification of four pome fruit viruses by one-tube pentaplex RT-PCR. Journal of Virological Methods 133 : 124-129. 10.1016/j.jviromet.2005.11.00216337013
4
Heo, S., H. R. Kim, and H. J. Lee. 2019. Development of a quantitative real-time nucleic acid sequence based amplification (NASBA) assay for early detection of Apple scar skin viroid. Plant Pathol Journal 35 : 164-171.
5
James, D., A. Varga, G. D. Jesperson, M. Navratil, D. Safarova, F. Constable, M. Horner, K. Eastwell, and W. Jelkmann. 2013. Identification and complete genome analysis of a virus variant or putative new foveavirus associated with apple green crinkle disease. Archives of Virology 158 : 1877-1887. 10.1007/s00705-013-1678-723553453
6
Kim, H. R., D. H. Lee, J. S. Kim, S. H. Lee, K. O. Yun, and H. I. Jang. 2004. Occurrence and Bioassay of Apple scar skin viroid (ASSVd) in Korea. Poster session presented at Korean Society for Horticultural Science, Horticultural Science and Technology 22 : 38.
7
Korean Statistical Information Service (KOSIS). 2018. http:// kosis.kr/index/index.do
8
Kumar, S., L. Singh, R. Ram, A. A. Zaidi, and V. Hallan. 2014. Simultaneous detection of major pome fruit viruses and a viroid. Indian Journal of Microbiology 54 : 203-210. 10.1007/s12088-013-0431-y25320423PMC4188501
9
Kumar, S., G. Stecher, M. Li, C. Knyaz, and K. Tamura. 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35 : 1547-1549. 10.1093/molbev/msy09629722887PMC5967553
10
Mackay, I. M. 2004. Real-time PCR in the microbiology laboratory. Clinical Microbiology and Infection 10 : 190-212. 10.1111/j.1198-743X.2004.00722.x15008940
11
Magome, H., N. Yoshikawa, T. Takahashi, T. Ito, and T. Miyakawa. 1997. Molecular variability of the genomes of capilloviruses from apple, Japanese pear, European pear and citrus trees. Phytopathology 87 : 389-396. 10.1094/PHYTO.1997.87.4.38918945117
12
Malandraki, I., D. Beris, I. Isaioglou, A. Olmos, C. Varveri, and N. Vassilakos. 2017. Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR. PLoS ONE 12 : e0180877. 10.1371/journal.pone.018087728749955PMC5547701
13
Maliogka, V., A. Minafra, P. Saldarelli, A. Ruiz-García, M. Glasa, N. Katis, and A. Olmos. 2018. Recent advances on detection and characterization of fruit tree viruses using high-throughput sequencing technologies. Viruses 10 : 436. 10.3390/v1008043630126105PMC6116224
14
Menzel, W., W. Jelkmann, and E. Maiss. 2002. Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control. Journal of Virological Methods 99 : 81-92. 10.1016/S0166-0934(01)00381-0
15
Nemeth, M. 1986. Virus, Mycoplasma and Rickettsia Diseases of Fruit Trees. Nijhoff and Dr. W. Junk Publishers. p. 750.
16
Nickel, O. and T. V. M. Fajardo. 2014. Detection of viruses in apples and pears by real time RT-PCR using 5'-hydrolysis probes. Journal of Plant Pathology 96 : 207-213.
17
Nickel, O., W. Jelkmann, and G. B. Kuhn. 1999. Occurrence of Apple stem grooving capillovirus in Santa Catarina, Brazil, detected by RT-PCR. Fitopatologia Brasileira 24 : 444-446.
18
Winkowska, L., L. Grimova, and P. Rysanek. 2016. Quantitative detection of four pome fruit viruses in apple trees throughout the year. Phytopathologia Mediterranea 55 : 207-224.
Information
  • Publisher :The Korean Society of Crop Science
  • Publisher(Ko) :한국작물학회
  • Journal Title :The Korean Journal of Crop Science
  • Journal Title(Ko) :한국작물학회지
  • Volume : 65
  • No :4
  • Pages :496-507
  • Received Date : 2020-06-13
  • Revised Date : 2020-09-18
  • Accepted Date : 2020-09-27
  • DOI :https://doi.org/10.7740/kjcs.2020.65.4.496
Journal Informaiton The Korean Journal of Crop Science The Korean Journal of Crop Science
Online Submission
  • NRF
  • KOFST
  • KISTI Current Status
  • KISTI Cited-by
  • crosscheck
  • orcid
  • open access
  • ccl
Journal Informaiton Journal Informaiton - close