Detection Of Three Important Aquatic Viruses Using Real-time PCR Assay

To establish a specific,sensitive method of TaqMan-based real time PCR assay for the rapid detection of channel catfish virus,the ORF8 gene of Channel catfish virus was down-loaded from Genbank and the specific primers and probes were designed.The primers and probes as well as the reaction condition were optimized to improve the sensitivity and specificity of the assay.The specificity,sensitivity,reproducibility of the method were estimated and a standard curve was prepared.It was found that the specificity was high without any cross-reactions with KHV,SGIV,KNV,RSIV and other commonly encountered viruses from aquatic animals.A good linear correlation was demonstrated in the standard curve for the real-time PCR assay within the range from 3.3×10～3.3×10⁷ copies.A minimum of 33 positive plasmids could be detected, indicating a good sensitivity of the assay.The coefficients of variation were 0.7%and 1.2 %for the intra-assay and inter-assay tests respectively,indicating a good reliability.It took only 3 hours to complete the whole course of reaction including extraction of viral DNA and the real-time PCR.The results demonstrate that real-time PCR method can be used as an effective detection and quantification method for CCV and it is amenable to high-throughout assay for its specificity,sensitivity and rapidity.To establish a real-time PCR method for rapid detection of Ranavirus(family Iridoviridae) The MCP gene of Ranavirus were down-loaded from Genbank.The gene sequences of MCP were aligned using the biologic software and the specific primers and probe were designed in the conserved region.The primers,probe and the reactive condition were optimized to improve the sensitivity.The specificity,sensitivity, reproducibility of the method were estimated And establish a relatively quantitative standard curve.It was found that the specificity of this assay was high for member of Ranavirus(except for SGIV) without any cross-reactions with LCDV,RSIV,ISKNV, KHN and other commonly encountered viruses from aquatic animals.A good linear correlation was demonstrated in the standard curve for the real-time PCR assay.A minimum of 4.5×10^-3 pg/μl total DNA could be detected,indicating a good sensitivity of the assay.The coefficients of variance(CV) were 1.2%and 1.6%for the intra-assay and inter-assay tests respectively,indicating a good reliability.The results showed that this assay is a valuable complementary tool to the routine detection of Ranavirus.It has potential to apply in entry-exit inspection and quarantine.A real-time RT-PCR assay for fish nervous necrosis virus was developed.The specific primers and TaqMan probes were designed according to the highly conservative sequence of capsid protein(CP) gene of Viral nervous necrosis virus from Genbank. After the procedure of the real-time RT-PCR assay for fish nervous necrosis virus was optimized,the specificity,sensitivity,reproducibility of the method were estimated.It was found that the specificity of this assay was high without any cross-reactions with SVC,IPN,VHSV,GCHV,IHNV,and so on.A minimum of 1.2 pg/μl total RNA could be detected,indicating a good sensitivity of the assay.The coefficients of variance(CVs) were 0.9%and 1.5%for the intra-assay and inter-assay tests respectively,which indicated good reproducibility.It took only 4 hours to complete the whole course of reaction including extraction of viral RNA and the real-time RT-PCR.Detected by the real-time RT-PCR assay for fish nervous necrosis virus,it was found that forty specimen were positive among 500 clinical samples.Those results confirmed that the real-time RT-PCR assay for VNNV was a rapid,sensitive and specific method for the detection of viral nervous necrosis virus.