A one‐step molecular biology method for simple and rapid detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 strain

Abstract

Six reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primers designed against conserved regions of segment 6 (s6) gene were used for the detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 subtype. The entire amplification could be completed within 40 min at 62·3° C. The RT-LAMP showed higher sensitivity than reverse-transcription polymerase chain reaction (RT-PCR). The RNA detection limit was 10 copies µl⁻¹ for RT-LAMP assay and 100 copies µl⁻¹ for conventional RT-PCR. In specificity tests, no cross-reactivity was detected in other viruses from common aquatic animals. In addition, the reaction results can be visualized by using calcein fluorescent dye. Furthermore, a total of 86 samples were tested by RT-LAMP, RT-PCR and virus isolation. The results demonstrated that all 54 specimens identified as positive by virus isolation were also positive when detected by RT-LAMP. Seven out of 54 samples, however, were misidentified by RT-PCR. The RT-LAMP method is more accurate than conventional RT-PCR. The results indicate that RT-LAMP has potential as a simple and rapid diagnosis technique for the detection of GCRV HZ08 subtype infection.

Development of a VP38 recombinant protein‐based indirect ELISA for detection of antibodies against grass carp reovirus genotype II (iELISA for detection of antibodies against GCRV II)

“…Vaccination and pathogen surveillance are considered to be the most effective measures of protecting grass carp from this disease. At present, GCHD is mainly diagnosed by detecting of the virus specific nucleic acid, including PCR (Zeng, Wang, Wang, Zhang et al., ; Zeng, Wang, Wang, Shi et al., ; Zhang, Luo, Fang, & Wang, ), qPCR (Zeng et al., ), RT‐LAMP (Zeng, Wang, Wang, Zhang et al., ; Zeng, Wang, Wang, Shi et al., ) or NASBA‐ELISA (Zeng, Yao et al., ). Only a few of the immunological methods have been developed, and most are still for detecting virus detection (He et al., ; Jing et al., ; Wang et al., ; Zeng, Wang et al., ) There has been no feasible and effective method for the detection of the antibody level specific to GCRV II until now.…”

“…In recent years, the GCRV II was the major pathogen to induce GCHD in China (Pei et al, 2014;Zeng, Wang, Wang, Zhang et al, 2013;Zeng, Wang, Wang, Shi et al, 2013). Attempts to control GCRV infection was restrained by a lack of knowledge of the pathogenesis of the disease as well as of possible anti-viral therapeutics.…”

“…GCRV can be divided into three genotypes in China based on differences in genome sequences (Wang et al., ). Genotype II is associated with major outbreaks of grass carp hemorrhagic disease (Pei, Ke, Chen, & Zhang, ; Zeng, Wang, Wang, Shi, & Wu, ; Zeng, Wang, Wang, Zhang, & Wu, ). It is therefore important to specifically diagnose the grass carp hemorrhagic disease caused by genotype II GCRV.…”

Rapid detection of grass carp reovirus type 1 using RPA-based test strips combined with CRISPR Cas13a system

“…In addition, immunoblotting using specific antibodies is another method for virus detection (Jing et al, 2014), but it also suffers from the drawback of low sensitivity. Consequently, tools for detecting the GCRV genome, including polymerase chain reaction (PCR), real-time fluorescence quantitative PCR, or loop-mediated isothermal amplification (LAMP), have gained popularity because they are highly sensitive and specific in diagnosing GCRV (Zhang et al, 2010;Zeng et al, 2013;Zhang et al, 2013;Jing et al, 2014). Nonetheless, a main challenge with such technologies lies in their requirements of sophisticated equipment and trained professional manipulations, which hinders their practical application.…”

Phylogenetic analysis of newly isolated grass carp reovirus

“…To date, a number of various GCRV isolates have been isolated from diseased grass carp around the world, including GCRV 873, GCRV 875, GCRV HZ08, GCRV GD108, AGCRV and others (Fang et al 2002; Chi et al 2011; Ye et al 2012; Zeng et al 2013). These isolates are distinct not only in their levels of virulence and cell culture characteristics, but also in their antigenicity (Fang et al 2002; Mohd Jaafar et al 2008; Zhang et al 2010a).…”