Development of a rapid, sensitive and specific diagnostic assay for fish Aquareovirus based on RT-PCR

An improved RT-PCR assay for rapid and sensitive detection of grass carp reovirus

“…In this study, the sensitivity of detection could also be improved by combining easy column RNA extraction with segment 10 primed RT-PCR. Using this approach, a targeting sensitivity of 10 −3 pg was achieved compared with 10-100 pg of purified aquareovirus dsRNA in the method reported by Seng et al (2004) and 0.01 pg with the RT-PCR described by Guo et al (2008). In addition, the primers of segment 10, which amplify a fragment of about 500 bp, are sufficient for detection of all four isolates of GCRV rather than a single isolate, as reported by Li et al (1997).…”

“…Li et al (1997) developed previously a GCRV RT-PCR detection method based on segment 6 and segment 9, but their two primer pairs could only detect one of two GCRV isolates, GCRV-861; GCRV-873, the most virulent strain, could not be detected. More recently, Seng et al (2004) developed another RT-PCR detection method which could detect segment 6 from five species of aquareovirus including threadfin reovirus, guppy reovirus, grass carp reovirus, chum salmon reovirus and striped bass reovirus; however, this method is not GCRV specific and has limited specificity.…”

“…The first step involves extraction of total RNA from cells or tissue infected by virus, and the second step is cDNA-related RT-PCR amplification. For the first step, phenol:chloroform or the commercial RNA extracting reagent Trizol is used commonly (Li et al, 1997;Seng et al, 2004;Guo et al, 2008). However, this method is not only inconvenient for working with a large number of samples, but also decrease the RNA content of the samples by repeated extraction and precipitation steps.…”

“…Segment 6 of GCRV was used as a template in a previous report (Li et al, 1997;Seng et al, 2004). Therefore, to evaluate further the sensitivity of the RT-PCR detection strategy of targeting on S10 or S6, the amplification ability of the GCRV-S10F2/R and GCRV-S6F/R1 primer pairs (both yielding ∼500 bp amplicons) were compared.…”

“…Additionally, reverse transcription-polymerase chain reaction (RT-PCR) techniques have also been developed (Li et al, 1997;Seng et al, 2004;Guo et al, 2008). However, RT-PCR detection methods can be hampered by technical difficulties associated with efficient preparation of dsRNA templates and optimal genome segment priming or targeting.…”