Rapid detection and differentiation of carp oedema virus and cyprinid herpes virus‐3 in koi and common carp

Abstract

Carp oedema virus (CEV) and koi herpes virus (KHV) are of major concern to common carp breeders and koi enthusiasts worldwide. The viruses cause diseases that exhibit similar external signs; thus, it is difficult to distinguish between them clinically. In this study, we developed and optimized rapid and accurate single- and multiplex isothermal diagnostic tools, based on recombinase polymerase amplification (RPA), for detection and differentiation of CEV and KHV. The assays were combined with a lateral flow dipstick to enable visual detection of amplification products and simplify post-amplification analysis. Both CEV- and KHV-RPA assays were specific for their target virus. The lower detection limits of the assays were similar to those of established diagnostic PCR tests for the viruses. A sample preparation method was optimized to eliminate the need for total DNA extraction from fish tissues. The estimated time to perform these RPA assays, from receiving the sample to having a result, is 50 min, compared to 10 and 7 hr for CEV- and KHV-PCR tests, respectively. The assays can be performed in field situations to improve screening of fish and reduce spread of these viruses and thereby enhance the common carp and koi industries.

Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3)

“…Our results were similar to other studies using lateral flow assay strips for the rapid detection of KHV [ 30 , 31 ]. The latter based their diagnostic systems on the loop mediated isothermal amplification and the isothermal DNA amplification assay using recombinase polymerase amplification [ 30 , 31 ]. In contrast, we based our assay on a more common amplification technique, the PCR.…”

First Evidence of Carp Edema Virus Infection of Koi Cyprinus carpio in Chiang Mai Province, Thailand

“…The cumulative mortality rate in this case was 20%, which is not consistent with previous reports [15,16,23]. Mortality and morbidity rates unusually decrease when temperatures increase to 28 • C [26], which may explain the lower mortality observed in this case. Nevertheless, some previous reports indicate that KSD occurs at lower temperatures.…”

“…The most common behavioral abnormality of CEV infection in cyprinid fish is lying on the bottom of the ponds. Other clinical signs of infected fish include: lethargy, anorexia, swelling and necrotic gills, enophthalmos, and skin hemorrhages or skin ulceration [23][24][25][26]. Similar clinical signs were observed in the present episode.…”

“…However, because of the similarities of pathognomonic signs of viruses affecting in koi such as koi herpesvirus (KHV) and SVC, specific diagnostic procedures are required. Various CEV diagnostic assays have been established based on genetic detection of the virus by PCR [25,26]. Importantly, there have been no previous reports of KSD in Thailand until this date.…”

Review: a comprehensive summary of a decade development of the recombinase polymerase amplification

“…However, several research groups have studied RPA reaction temperatures that lie outside of the recommended range. 38,44,45,60,[62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78] The largest temperature range was tested between 15°C and 50°C; 62,64,69,70,76 and results indicated the marginal reaction temperature to produce a positive result should be greater than 30°C. [62][63][64]66,67,69,71,74,76,77 However, Sun et al 65 and Poulton and Webster 60 showed that temperature as low as 25°C could still generate a positive signal after RPA amplification and subsequent lateral flow strip detection.…”