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Abstract White spot syndrome virus (WSSV) , Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and and Taura syndrome virus (TSV ) are responsible for significant economic loss in the shrimp industry. In order to simultaneously and massively identify WSSV , IHHNV and TSV, three pair of primers and three TaqMan probes were designed and synthesized according to the conserved gene sequences of WSSV (AF369029) , IHHNV (AF218226) and TSV(NC003005) in GenBank. The reaction parameters such as the concentration of three pair of primers, three TaqMan probes and the reaction buffer were optimized to develop a multiplex real-time PCR assay for the rapid detection of WSSV , IHHNV and TSV. The multiplex real-time PCR assay was found to be specific and to be able to detect and differentiate WSSV , IHHNV and TSV, and no positive results were observed when nucleic acid from Vibrio and Streptococcus were used as multiplex real-time PCR templates. The developed multiplex real-time PCR assay was compared with that of routine PCR. The sensitivity of multiplex real-time PCR assay was 20000, 20 and 20000 template copies for WSSV , IHHNV and TSV respectively, and its sensitivity was 10, 1000 and 10 times higher than that of the routine PCR. The samples were examined using the multiplex real-time PCR repeatedly and the results indicated that the multiplex real-time PCR was reproducible. Different concentrations of WSSV , IHHNV and TSV when mixed together still could be identified by this assay, which implied the assay could be applied to clinical confirmation for simultaneous infection of WSSV , IHHNV and TSV. The multiplex real-time PCR results of 45 routine PCR positive samples showed that one specific amplified curve was displayed when shrimp was infected by only one of these three viral pathogens, whereas two or three specific amplified curves were displayed when shrimp was infected by two or three viral pathogens. The result indicated that multiplex real-time PCR was able to detect and differentiate the presence of each pathogen in infected clinical shrimp. This multiplex real-time PCR assay was a quick, sensitive, specific and quantitative tool for detection of WSSV , IHHNV and TSV, and will be useful for the control of WSS, IHHN and TS in shrimp.
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Received: 26 November 2007
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