An improved RT-PCR assay for rapid and sensitive detection of grass carp reovirus.
An improved simple, rapid and sensitive method for detecting grass carp reovirus (GCRV) based on RT-PCR was developed by combining an advanced RNA extraction technique and targeting segment 10 as a template. The results indicate that highly efficient RT-PCR amplification of GCRV genome segments can be obtained using column-extracted RNA as a template, which is suitable not only for full-length gene amplification up to a size of 1.5 kb, but also for partial genome detection. Moreover, by targeting the highly divergent segment 10, the sensitivity of RT-PCR detection is improved significantly; as little as 1.0 fg of the 515 bp S10 dsRNA can be detected by one-step RT-PCR amplification. Furthermore, this method exhibits good reproducibility and specificity, and no amplicons were observed when RNA fragments other than those from GCRV were used as templates. The entire detection process can be completed within 4-5h from RNA extraction, much faster than methods reported previously. Overall, the improved detection technique may be applied for rapid diagnosis of GCRV or other dsRNA viruses.
Zhang
Lanlan
L
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Luo
Qing
Q
Fang
Qin
Q
Wang
Yaping
Y
D000818:Animals; D002347:Carps / Q000821:virology*; D002853:Chromatography, Liquid / Q000379:methods; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012087:Reoviridae / Q000302:isolation & purification*; D012088:Reoviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014773:Virology / Q000379:methods*
D012367:RNA, Viral