Koi Herpesvirus (KHV) is a highly contagious viral disease which causes significant morbidity and mortality in common carp (Cyprinus carpio) and its ornamental domesticated form, koi carp . Although the virus is currently regarded as a DNA-virus belonging to family Herpesviridae , some reports have disputed this classification and have renamed the virus as Carp Nephritis and Gill Necrosis Virus, CNGV . More recently, reports based on morphology and genetics have demonstrated strong evidence that KHV is indeed a herpesvirus .
The international trade in live fish is arguably the most effective dispersal pathway of fish diseases through incidental movement of pathogenic organisms .With respect to koi, exhibitions and national and international trading have facilitated the rapid global spread of KHV. The disease struck koi population in the USA and Israel in 1998 and spread rapidly ; it has been reported in Germany , Korea [7, 8], Indonesia , Japan , South Africa, and Thailand (unpublished data).
Clinical signs of KHV are often non-specific and mortality may occur rapidly. Discoloration and severe necrosis of the gills is the most consistent sign of infection, with disorientation and erratically swimming prior to death, which can occur within 24–48 hours after the onset of clinical signs [11, 12]. KHV has caused considerable economic losses in both the koi and carp culture industries: to fish breeders, retailers and hobbyists impacted by the cumulative mortalities associated with outbreaks [4, 2]. There is a clear need for a reliable, rapid diagnostic procedure for the detection of KHV infection.
Rapid virological diagnosis through isolation of the virus has proven difficult and time consuming. A far more efficient approach is nucleic acid amplification; one of the most valuable tools in virtually all life science fields . One of the most widely used techniques is the polymerase chain reaction (PCR) which uses heat denaturation of double-stranded DNA products to promote the next round of DNA synthesis [14, 15]. A widely used PCR assay for KHV was developed , and a second PCR assay for KHV has been described . A real-time TaqMan PCR assay for KHV has also been developed to detect and quantify KHV DNA in infected tissues . While these PCR techniques have significantly increased our ability to detect KHV infection in koi and common carp, their requirement for a high precision thermacycler has prevented their widespread use in private clinics, for example, as a routine diagnostic tool.
A novel nucleic acid amplification method, loop-mediated isothermal amplification (LAMP), has been developed that does not require a theramcycler. LAMP relies instead on autocycling strand displacement DNA synthesis by a Bst DNA polymerase, to amplify DNA with high specificity, efficiency, and speed under isothermal conditions [13, 18, 19]. LAMP requires two specially designed inner and two outer primers to improve specificity [20, 21]; if two additional 'loop' primers are added, the reaction time can be halved . The amplification products are stem-loop DNA structures with several inverted repeats of the target, and cauliflower-like structures comprising multiple loops . In the present study, we used a LAMP technique for diagnosis of KHV, and evaluated its sensitivity, specificity, and applicability.