Development of a sensitive and quantitative assay for spring viremia of carp virus based on real-time RT-PCR

Zhiqin Yue, Yong Teng, Chengzhu Liang, Xiayang Xie, Biao Xu, Laihua Zhu, Zhiwen Lei, Junqiang He, Zongxiao Liu, Yulin Jiang, Hong Liu, Qiwei Qin

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

A one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) using a TaqMan probe to quantitatively detect spring viremia of carp virus (SVCV) is described. In this assay, a pair of primers amplifying an 81-bp DNA fragment and a TaqMan probe was designed targeting the conserved region at the SVCV glycoprotein (G) gene. To avoid the disadvantages arising from plasmids, an extension adding a T7 phage polymerase promoter to the 5′ end of the antisense primer was carried out to obtain viral cRNA. Standardized cycle threshold (Ct) values for 10-fold serial dilutions of SVCV cRNA were achieved by real-time RT-PCR and used to create standard curves. A regression line between the mean Ct values and viral template concentrations over a 1:107 dilution range with an r2 value (0.9916) and a slope (-3.36) and the coefficient of variation (intra- or inter-assay is <2% and <4%, respectively) indicated that the assay was highly reproducible. The assay was specific to SVCV and there was no cross-reactivity with other fish viruses (viral hemorrhagic septicemia virus, VHSV; infectious pancreatic necrosis virus, IPNV; grass carp reovirus, GCRV; epizootic haematopoietic necrosis virus, EHNV). The standard curve allows precise absolute quantitation and shows that the detection limit of the assay is 40 copies of the viral RNA. This one-step RT-PCR assay was evaluated using 60 clinical common carp samples collected during the year 2006, indicating such technology offers considerable advantages over conventional RT-PCR methods in current routine use for SVCV surveillance. This is the first report of the development of a one-step TaqMan® RT-PCR for SVCV detection.

Original languageEnglish (US)
Pages (from-to)43-48
Number of pages6
JournalJournal of Virological Methods
Volume152
Issue number1-2
DOIs
StatePublished - Sep 1 2008

Fingerprint

Carps
Viremia
Reverse Transcription
Viruses
Polymerase Chain Reaction
Infectious pancreatic necrosis virus
Complementary RNA
Novirhabdovirus
Bacteriophage T7
Viral RNA
Limit of Detection
Glycoproteins
Fishes
Plasmids
Necrosis
Technology
DNA

Keywords

  • Diagnostic assay
  • Real-time RT-PCR
  • SVCV
  • TaqMan probe

ASJC Scopus subject areas

  • Virology

Cite this

Development of a sensitive and quantitative assay for spring viremia of carp virus based on real-time RT-PCR. / Yue, Zhiqin; Teng, Yong; Liang, Chengzhu; Xie, Xiayang; Xu, Biao; Zhu, Laihua; Lei, Zhiwen; He, Junqiang; Liu, Zongxiao; Jiang, Yulin; Liu, Hong; Qin, Qiwei.

In: Journal of Virological Methods, Vol. 152, No. 1-2, 01.09.2008, p. 43-48.

Research output: Contribution to journalArticle

Yue, Z, Teng, Y, Liang, C, Xie, X, Xu, B, Zhu, L, Lei, Z, He, J, Liu, Z, Jiang, Y, Liu, H & Qin, Q 2008, 'Development of a sensitive and quantitative assay for spring viremia of carp virus based on real-time RT-PCR', Journal of Virological Methods, vol. 152, no. 1-2, pp. 43-48. https://doi.org/10.1016/j.jviromet.2008.05.031
Yue, Zhiqin ; Teng, Yong ; Liang, Chengzhu ; Xie, Xiayang ; Xu, Biao ; Zhu, Laihua ; Lei, Zhiwen ; He, Junqiang ; Liu, Zongxiao ; Jiang, Yulin ; Liu, Hong ; Qin, Qiwei. / Development of a sensitive and quantitative assay for spring viremia of carp virus based on real-time RT-PCR. In: Journal of Virological Methods. 2008 ; Vol. 152, No. 1-2. pp. 43-48.
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AU - Xie, Xiayang

AU - Xu, Biao

AU - Zhu, Laihua

AU - Lei, Zhiwen

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AU - Jiang, Yulin

AU - Liu, Hong

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