Detection of spring viraemia of carp virus (SVCV) by loop-mediated isothermal amplification (LAMP) in koi carp, Cyprinus carpio L.
Spring viraemia of carp virus (SVCV) is a rhabdovirus associated with systemic illness and mortality in cyprinids. Several diagnostic tests are available for detection of SVCV. However, most of these tests are time consuming and are not well adapted for field-based diagnostics. In this study, a diagnostic tool for SVCV detection based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been developed. Based on the nucleotide sequence of the glycoprotein (G) gene of SVCV North Carolina (NC) isolate, four sets (each set containing two outer and two inner) of primers were designed. Temperature and time conditions were optimized to 65 degrees C and 60 min, respectively, for LAMP and RT-LAMP using one primer set. In vitro specificity was evaluated using four different strains of fish rhabdoviruses and RT-LAMP was found to be specific to SVCV. Serial dilutions of SVCV NC isolate was used to evaluate the in vitro sensitivity of RT-LAMP. Sensitivity of the assays was similar to RT-PCR and detected SVCV even at the lowest dilution of 10(1) TCID50 mL(-1). The ability of RT-LAMP to detect SVCV from infected carp was also tested and the assay detected SVCV from all infected fish. The isothermal temperature requirements, high specificity and sensitivity, and short incubation time of the RT-LAMP assay make it an excellent choice as a field diagnostic test for SVCV.
Shivappa
R B
RB
College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA. raghunath_shivappa@ncsu.edu
Savan
R
R
Kono
T
T
Sakai
M
M
Emmenegger
E
E
Kurath
G
G
Levine
J F
JF
D000818:Animals; D001483:Base Sequence; D002347:Carps / Q000821:virology*; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000737:chemistry; D005393:Fish Diseases / Q000175:diagnosis / Q000821:virology*; D021141:Nucleic Acid Amplification Techniques / Q000662:veterinary*; D018353:Rhabdoviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D012680:Sensitivity and Specificity; D018116:Vesiculovirus / Q000235:genetics / Q000302:isolation & purification*; D014759:Viral Envelope Proteins / Q000235:genetics*
D017931:DNA Primers; D014759:Viral Envelope Proteins