Development of a sensitive and quantitative assay for spring viremia of carp virus based on real-time RT-PCR.
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 1:10(7) dilution range with an r(2) 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.
Yue
Zhiqin
Z
Shandong Entry-Exit Inspection and Quarantine Bureau, No. 1 Tuandao Er Road, Qingdao 266002, China. yuezhiqin@126.com
Teng
Yong
Y
Liang
Chengzhu
C
Xie
Xiayang
X
Xu
Biao
B
Zhu
Laihua
L
Lei
Zhiwen
Z
He
Junqiang
J
Liu
Zongxiao
Z
Jiang
Yulin
Y
Liu
Hong
H
Qin
Qiwei
Q
D000818:Animals; D002347:Carps / Q000821:virology*; D002460:Cell Line; D005393:Fish Diseases / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012209:Rhabdoviridae / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D012680:Sensitivity and Specificity; D014766:Viremia / Q000662:veterinary* / Q000821:virology