Nervous necrosis virus (NNV) is a single-stranded RNA virus, belonging to the genus
Betanodavirus within the family
Nodaviridae whose genome consists of two molecules of RNA: RNA1 and RNA2. This virus is the pathogen that causes the viral nervous necrosis (VNN) in many species of fish. It can lead to large-scale death of the larvae and juveniles and the cumulative mortality can be as high as 100 % within 1 week after infection [
1]. This virus is widely distributed. There have been reports about its epidemic in Asia, Australia, North America, and Europe [
2,
3]. NNVs are divided into four genotypes, including barfin flounder nervous necrosis virus (BFNNV), red-spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus (SJNNV) and tiger puffer nervous necrosis virus (TPNNV). Among them, RGNNV is the most popular one. Currently, there have been no methods available for effective treatment of VNN caused by these types of virus. Thus, it is of extreme importance to establish a rapid and highly sensitive method for detection of these pathogens for prevention and control of this disease.
Currently, there have been several methods available for detection of NNV, including enzyme-linked immunosorbent assay (ELISA) [
4], indirect fluorescent antibody test (IFAT) [
5], cell culture [
6] and molecular biology-based methodologies [
7‐
13]. Among them, the molecular biology-based methodologies are more rapid and accurate and thus, the most widely used methods applied in the detection of NNV in fish samples. The molecular biology-based methodologies include the conventional reverse transcription-polymerase chain reaction (RT-PCR) [
7,
8], loop-mediated isothermal amplification (LAMP) [
9‐
11], real-time quantitative RT-PCR (qRT-PCR) [
12,
13] etc. While these methodologies have been used in detection of NNV, each of them has certain limitations and shortcomings. For instance, the sensitivity for conventional RT-PCR is not high enough as its lowest limit of detection (LLOD) is as high as 10
2 copies and thus, it does not meet the requirement for prevention and control of NNV. Although qRT-PCR is highly sensitive, this method requires the expensive equipment and reagents and can’t be used on the spot. While LAMP is relatively sensitive and can be used on the spot, its specificity was relatively low due to the use of staining dyes for color development of the amplified products. Cross-priming isothermal amplification (CPA) is a currently developed, nucleic acid-based amplification method which is rapid and highly effective [
14]. With this method, at least one cross primer is used, making the sequences of the amplified product to form semi-loop single-stranded structure or branched structures. Under the action of
Bst DNA polymerase which possesses the strand displacement activity, the targeted DNA sequence is continually amplified at a constant temperature. This technology has been successfully applied in the detection of many types of bacteria and virus, including
Mycobacterium tuberculosis [
15],
Enterobacter sakazakii [
16],
Acidovorax citrulli [
17], fowl adenovirus [
18], and white spot syndrome virus [
19] etc. In term of the analysis of nucleic acid-based amplified products, lateral flow dipstick (LFD) detection technology has been widely applied because it has a characteristic of rapid reaction and can be easily carried [
20‐
22]. The principle of LFD is as follow: the amplified nucleic acid product doubly labeled with FITC and Biotin is drop-wise added on the dipstick, which interacts with the anti-biotin antibody- carrying colloidal gold and this complex is migrated with the liquid flow along the dipstick to the detection line where it is captured by anti-FITC antibody and aggregated, finally forming a red and macroscopical strip on the detection line. CPA coupled with LFD (CPA-LFD) may allow achieving the rapid and effective detection of pathogens on the spot [
20,
23].
In this study, we established a cross-priming-isothermal amplification coupled with lateral flow dipstick method for specific detection of RGNNV and applied this method in detection of RGNNV in fish samples. The establishment of this method has provided a convenient way for rapid detection of RGNNV on the spot for those laboratories that have only simple equipment or aquaculture farms.