Sensitive and rapid detection of infectious pancreatic necrosis virus by reverse transcription loop mediated isothermal amplification.
A new molecular diagnostic assay was developed for rapid and sensitive diagnosis of infectious pancreatic necrosis virus (IPNV) by using a one step, one tube reverse transcription loop-mediated isothermal amplification (RT-LAMP). A set of six LAMP primers was designed to amplify the target RNA by incubation with Bst DNA polymerase plus reverse transcriptase and the reaction was optimised at 65 degrees C for 60 min. Three different methods for detection of the amplified product by naked eye gave identical results to gel electrophoresis, which was run for confirmation. Negative results obtained with RNA from four other fish viruses confirmed the specificity of the test. The IPNV-RT-LAMP assay demonstrated superior analytical sensitivity compared to conventional RT-PCR conducted according to published methods (1:10(12) dilution of RNA extracted from an IPNV-infected cell culture supernatant vs. 1:10(6) for the conventional RT-PCR). The feasibility of the RT-LAMP assay for detection of IPNV RNA in clinical specimen was authenticated using kidney tissue samples from experimentally IPNV-infected Atlantic salmon (Salmo salar) post-smolts. The results suggest that the RT-LAMP is a rapid and highly sensitive diagnostic assay for IPNV which lends itself well to use in aquaculture health management and disease control.
Clinic for Fish and Reptiles, Faculty of Veterinary Medicine, University of Munich, Munich, Germany.
Hatem
H
Soliman
Paul J
PJ
Midtlyng
Mansour
M
El-Matbouli
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018061:Infectious pancreatic necrosis virus / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020317:Salmo salar; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
D017931:DNA Primers