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

J Fish Biol. 2013 May;82(5):1545-55. doi: 10.1111/jfb.12088. Epub 2013 Apr 16.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carps / virology*
  • Cell Line
  • Kidney / cytology
  • Nucleic Acid Amplification Techniques / methods*
  • Reoviridae / genetics
  • Reoviridae / isolation & purification*
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Sensitivity and Specificity
  • Virus Cultivation / methods