A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cyprinid Herpesvirus 2 in Gibel Carp (Carassius auratus gibelio)

Abstract

A rapid and sensitive loop-mediated isothermal amplification (LAMP) assay for Cyprinid herpesvirus 2 (CyHV-2) detection in gibel carp was developed. Following cloning and sequencing of the putative DNA helicase gene of CyHV-2 isolate from China, a set of four specific primers was designed based on the sequence. The MgCl2 concentration and the reaction temperature were optimized to 6 mM, 64°C, respectively. LAMP products were detected by visual inspection of a color change due to addition of SYBR Green I stain. The specificity and sensitivity of the LAMP assay were determined. No cross-reaction was observed with other fish DNA viruses including eel herpesvirus, koi herpesvirus, and Chinese giant salamander iridovirus. The LAMP assay was found to be equally sensitive as nested PCR. A comparative evaluation of 10 fish samples using LAMP and nested PCR assays showed an overall correlation in positive and negative results for CyHV-2. These results indicate that the LAMP assay is simple, sensitive, and specific and has a great potential use for CyHV-2 detection in the laboratory and field.

Identification and rapid diagnosis of the pathogen responsible for haemorrhagic disease of the gill of Allogynogenetic crucian carp

“…In the current study, a pair of loop primers: CyHV-LF and CyHV-LB were supplemented in the LAMP assay for CyHV-2 detection, which shortened the reaction time to 30 min and enhanced reaction efficiency. In addition, in the previous study, the DNA template used for CyHV-2 detection was extracted from the diseased fish by a commercial DNA extraction kit (Zhang et al, 2014). However, in the current study, the template DNA was simply prepared by boiling in a water bath for 5 min, which is more economical and time-efficient.…”

“…Virus isolation may be difficult (Gilad et al, 2002) and detection of viral pathogens in host cells depends upon sensitive cell lines, subculture of cells and virus, and is time consuming. As CyHV-2 is very difficult to culture in cells (Goodwin et al, 2009;Xu et al, 2013), molecular diagnostics including conventional PCR, nested PCR, and real-time PCR assays are primarily used for the detection and quantification of this virus (Goodwin et al, 2009;Zhang et al, 2014;Zhou et al, 2013). These molecular methods have become the "gold standard" for rapid diagnosis.…”

“…An assay fitting this description is LAMP, which can be performed using very simple laboratory equipment such as a water bath or heating block. Recently, Zhang et al have established a LAMP assay for CyHV-2 detection (Zhang et al, 2014). This LAMP assay was performed under isothermal conditions (64 • C) for 1 h with a pair of inner primers and a pair of outer primers.…”

Spring viraemia of carp virus: recent advances

“…Moreover, it requires uncomplicated and inexpensive equipment that can be easily manipulated at fish farms. This assay has been successfully applied for disease control in aquaculture (Khunthong et al, 2013;Zhang et al, 2014). Two studies have used RT-LAMP to detect SVCV based on nucleotide sequences of the G and M genes (Shivappa et al, 2008;Liu et al, 2008b).…”

Virus genomes and virus-host interactions in aquaculture animals

“…In recent years, after cyprinid herpesvirus disease became widely reported in the world, (especially in China) [145][146][147], numerous studies on cyprinid herpesviruses and identification of immune-related genes have been conducted [148][149][150]. It is envisaged, therefore, that new insight and better understanding of these cyprinid herpesviruses will emerge and lead to efficient antiviral approaches being developed in the near future.…”