Location: Aquatic Animal Health ResearchTitle: A one-step molecular biology method for simple and rapid detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 strain Author
Submitted to: Journal of Fish Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/30/2013
Publication Date: 6/30/2013
Publication URL: http://handle.nal.usda.gov/10113/60501
Citation: Zeng, W., Wang, Q., Wang, Y., Xu, D., Wu, S. 2013. A one-step molecular biology method for simple and rapid detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 strain. Journal of Fish Biology. 82:1545-1555. Interpretive Summary: Currently, loop-mediated isothermal amplification (LAMP) has been developed successfully to diagnose many viruses from aquatic animals. The use of LAMP for detecting grass carp reovirus (GCRV) has not been reported. The objective of this study was to develop a rapid and simple assay to detect GCRV for aquaculture. A simple one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for GCRV detection was developed and validated. In this diagnostic protocol, the reaction was carried out in a single tube and completed in 40 min. The RT-LAMP showed higher sensitivity and specificity than the reverse-transcription polymerase chain reaction. The results in this study demonstrate that the RT-LAMP assay is a valuable technique for early detection and diagnosis of GCRV and will contribute to the prevention and control of GCRV infections.
Technical 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 62.3º C. The RT-LAMP showed higher sensitivity than reverse-transcription polymerase chain reaction (RT-PCR). The RNA detection limit was 10 copies ul-1 for RT-LAMP assay and 100 copies ul-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.