Hostname: page-component-6587cd75c8-ptrpl Total loading time: 0 Render date: 2025-04-24T06:57:20.562Z Has data issue: false hasContentIssue false
  • English
  • Français

Real-time PCR assay for sensitive organ detection and epidemic investigation of Turbot reddish body iridovirus

Published online by Cambridge University Press:  24 April 2009

Wu Cheng-Long ,
Shi Cheng-Yin ,
Huang Jie  and
Kong Xiao-Yu
Wu Cheng-Long
Affiliation:
Key Laboratory for Sustainable Utilization of Marine Fisheries Resource, Ministry of Agriculture; Yellow Sea Fisheries Research Institute, Qingdao 266071, China Key Laboratory of Mariculture, Ministry of Education; Ocean University of China, Qingdao 266003, China
Shi Cheng-Yin*
Affiliation:
Key Laboratory for Sustainable Utilization of Marine Fisheries Resource, Ministry of Agriculture; Yellow Sea Fisheries Research Institute, Qingdao 266071, China
Huang Jie
Affiliation:
Key Laboratory for Sustainable Utilization of Marine Fisheries Resource, Ministry of Agriculture; Yellow Sea Fisheries Research Institute, Qingdao 266071, China
Kong Xiao-Yu
Affiliation:
Key Laboratory of Mariculture, Ministry of Education; Ocean University of China, Qingdao 266003, China
*
*Corresponding author. E-mail: shicy@ysfri.ac.cn
Get access Rights & Permissions [Opens in a new window]

Abstract

A rapid and sensitive real-time polymerase chain reaction (PCR) assay coupled with SYBR Green I chemistry was developed for the quantitative detection of Turbot reddish body iridovirus (TRBIV) isolated from farmed turbot (Scophthalmus maximus). A 152 bp DNA fragment from the TRBIV major capsid protein (MCP) gene was involved in the real-time PCR (RT-PCR) assay using the Roter Gene 3000 sequence detection system. The PCR mixture contained a fluorescent dye, SYBR Green I, which exhibited fluorescence enhancement when bound to double-stranded (ds) DNA. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. The positive control plasmid, pUCm-T/TRBIV MCP, containing the target sequence, was quantified to make a standard curve for sample detection after serial tenfold dilution. Linear coefficient correlations between the cycle threshold (CT) value and logarithmic positive plasmid concentration were close to one (R2=0.9952) and the detection limit of the assay was 102 copies of positive plasmids. The quantitative detection of virus in different tissues from TRBIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (5.23×106 and 2.18×106 viral genome copies/mg tissue, respectively), while no viral DNA was detected in the muscular tissue. The molecular epidemic investigation of TRBIV showed that many cultured turbots were infected and TRBIV has become epidemic in turbot farms located along the Shandong peninsula. The virus number varied from 1.27×102 to 2.33×106 viral genome copies/mg tissue in spleens of infected turbot. These results suggest that the RT-PCR assay reported here can be used as a rapid, sensitive and quantitative method for TRBIV.

Keywords

Turbot reddish body iridovirus (TRBIV)real-time PCRSYBR Green Iquantitative detectionsensitive organ
Type
Research Papers
Information
Chinese Journal of Agricultural Biotechnology , Volume 6 , Issue 1 , April 2009 , pp. 61 - 67
Copyright
Copyright © China Agricultural University 2009

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Article purchase

Temporarily unavailable

References

Caipang, CMA, Hirono, I and Aoki, T (2003) Development of a real-time PCR assay for the detection and quantification of red seabream iridovirus (RSIV). Fish Pathology 38: 1–7.CrossRefGoogle Scholar
Caipang, CMA, Haraguchi, I, Ohira, T, Hirono, I and Aoki, T (2004) Rapid detection of a fish iridovirus using loop-mediated isothermal amplification (LAMP). Journal of Virological Methods 121: 155–161.CrossRefGoogle ScholarPubMed
Chao, CB, Yang, SC, Tsai, HY, Chen, CY and Huang, HT (2002) A nested PCR for the detection of grouper iridovirus in Taiwan (TGIV) in cultured hybrid grouper, giant seaperch, and largemouth bass. Journal of Aquatic Animal Health 14: 104–113.2.0.CO;2>CrossRefGoogle Scholar
Choi, SK, Kwon, SR, Nam, YK, Kim, SK and Kim, KH (2006) Organ distribution of red sea bream iridovirus (RSIV) DNA in asymptomatic yearling and fingerling rock bream (Oplegnathus fasciatus) and effects of water temperature on transition of RSIV into acute phase. Aquaculture 256: 23–26.CrossRefGoogle Scholar
Deng, M, He, JG, Zuo, T, et al. (2000) Infectious spleen and kidney necrosis virus (ISKNV) from Siniperca chuatsi: development of a PCR detection method and the new evidence of iridovirus. Chinese Journal of Virology 16(4): 365–369 (in Chinese).Google Scholar
Dhar, AK, Roux, MM and Klimpel, KR (2001) Detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR Green chemistry. Journal of Clinical Microbiology 39(8): 2835–2845.CrossRefGoogle ScholarPubMed
Go, J, Lancaster, M, Deece, K, Dhungyel, O and Whittington, R (2006) The molecular epidemiology of iridovirus in Murray cod (Maccullochella peelii) and dwarf gourami (Colisa lalia) from distant biogeographical regions suggests a link between trade in ornamental fish and emerging iridoviral diseases. Molecular and Cellular Probes 20: 212–222.CrossRefGoogle ScholarPubMed
Goldberg, TL, Coleman, DA, Grant, EC, Inendino, KR and Philipp, DP (2003) Strain variation in an emerging iridovirus of warm-water fishes. Journal of Virology 77(16): 8812–8818.CrossRefGoogle Scholar
Huang, CH, Zhang, XB, Gin, KYH and Qin, QW (2004) In situ hybridization of a marine fish virus, Singapore grouper iridovirus with a nucleic acid probe of major capsid protein. Journal of Virological Methods 117: 123–128.CrossRefGoogle ScholarPubMed
Jeong, JB, Park, KH, Kim, HY, et al. (2004) Multiplex PCR for the diagnosis of red sea bream iridovirus isolated in Korea. Aquaculture 235: 139–152.CrossRefGoogle Scholar
Kim, YJ, Jung, SJ, Choi, TJ, Kim, HR, Rajendran, KV and Oh, MJ (2002) PCR amplification and sequence analysis of irido-like virus infecting fish in Korea. Journal of Fish Diseases 25: 121–124.CrossRefGoogle Scholar
Kurita, J, Nakajima, K, Hirono, I and Aoki, T (1998) Polymerase chain reaction (PCR) amplification of DNA of red sea bream iridovirus (RSIV). Fish Pathology 33: 17–23.CrossRefGoogle Scholar
Mackay, IM, Arden, KE and Nitsche, A (2002) Real-time PCR in virology. Nucleic Acids Research 30(6): 1292–1305.CrossRefGoogle ScholarPubMed
Oshima, S, Hata, JI, Segawa, C, Hirasawa, N and Yamashita, S (1996) A method for direct DNA amplification of uncharacterized DNA viruses and for development of a viral polymerase chain reaction assay: Application to the red sea bream iridovirus. Analytical Biochemistry 242: 15–19.CrossRefGoogle Scholar
Oshima, S, Hata, J, Hirasawa, N, et al. (1998) Rapid diagnosis of red sea bream iridovirus infection using the polymerase chain reaction. Diseases of Aquatic Organisms 32: 87–90.CrossRefGoogle ScholarPubMed
Pellissier, F, Glogowski, CM, Heinemann, SF, Ballivet, M and Ossipow, V (2006) Lab assembly of a low-cost, robust SYBR Green buffer system for quantitative real-time polymerase chain reaction. Analytical Biochemistry 350(2): 310–312.CrossRefGoogle ScholarPubMed
Shi, CY, Wang, YG, Yang, SL, Huang, J and Wang, QY (2004) The first report of an iridovirus-like agent infection in farmed turbot, Scophthalmus maximus, in China. Aquaculture 236: 11–25.CrossRefGoogle Scholar
Shi, CY, Wang, YG, Huang, J and Wang, QY (2005) PCR amplification and sequence analysis of major capsid protein gene of turbot reddish body iridovirus (TRBIV). Journal of Fishery Sciences of China 12(5): 588–593 (in Chinese).Google Scholar
van Elden, LJR, Nijhuis, M, Schipper, P, Schuurman, R and van Loon, AM (2001) Simultaneous detection of influenza viruses A and B using real-time quantitative PCR. Journal of Clinical Microbiology 39(1): 196–200.CrossRefGoogle Scholar
Wang, XW, Ao, JQ, Li, QG and Chen, XH (2006) Quantitative detection of a marine fish iridovirus isolated from large yellow croaker, Pseudosciaena crocea, using a molecular beacon. Journal of Virological Methods 133: 76–81.CrossRefGoogle ScholarPubMed
Yuan, L, Zhang, XH, Chang, MX, Jia, CS, Hemmingsen, SM and Dai, HP (2007) A new fluorescent quantitative PCR-based in vitro neutralization assay for white spot syndrome virus. Journal of Virological Methods 146: 96–103.CrossRefGoogle ScholarPubMed