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A one‐step molecular biology method for simple and rapid detection of grass carp Ctenopharyngodon idella reovirus ( GCRV ) HZ08 strain
Author(s) -
Zeng W. W.,
Wang Q.,
Wang Y. Y.,
Xu D. H.,
Wu S. Q.
Publication year - 2013
Publication title -
journal of fish biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.672
H-Index - 115
eISSN - 1095-8649
pISSN - 0022-1112
DOI - 10.1111/jfb.12088
Subject(s) - grass carp , loop mediated isothermal amplification , reverse transcription loop mediated isothermal amplification , biology , detection limit , microbiology and biotechnology , reverse transcription polymerase chain reaction , real time polymerase chain reaction , reverse transcriptase , polymerase chain reaction , virology , gene , gene expression , dna , chromatography , fish <actinopterygii> , fishery , genetics , chemistry
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.