An assay using a single-tube, 1-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) was established for the simultaneous detection of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV). Three primer sets, 9195 F/9992 R, 94 F2/R2, and ITS F/28S R, were mixed at a ratio of 3:1:1 to amplify specific fragments of the TSV, WSSV, and Penaeus vannamei genome, respectively, in the RT-PCR reaction. Shrimp samples were experimentally infected with WSSV and TSV. PCR-amplified products detected in the nucleic acid extraction of shrimp pleopods produced 4 kinds of results. With no virus infection, 1 fragment of 892 base pairs (bp) was amplified from a ribosomal RNA gene by primer set ITS F/28S R as an internal control. In samples only infected by WSSV or TSV, 2 fragments could be seen: either from WSSV (530 bp) plus the internal control or TSV (231 bp) plus the internal control, respectively. In cases of co-infection with both viruses, all 3 amplified products were detected simultaneously. This study is the first report of Penaeus vannamei specimens co-infected with WSSV and TSV being detected using a PCR method via experimental infection.
KEY WORDS: RT-PCR · WSSV · TSV · Penaeid shrimp
Resale or republication not permitted without written consent of the publisherDis Aquat Org 50: [9][10][11][12] 2002 wide host range among crustaceans (Flegel 1997) and distinctive clinical signs (white spots) in penaeid shrimps. The entire sequence of the double-stranded, circular DNA genome has been determined (van Hulten et al. 2001). TSV is a single-stranded RNA virus that causes serious disease in the PL, juvenile and adult stages of P. vannamei exclusively (Lightner & Redman 1998).Reverse transcription (RT) and polymerase chain reaction (PCR) technologies have proven to be powerful diagnostic tools for shrimp viral infections and for detection of viral reservoirs in asymptotic carriers. Methods for PCR diagnosis have been published for WSSV (Kimura et al. 1996, Lo et al. 1996, Takahashi et al. 1996, Kim et al. 1998, Tapay et al. 1999 and TSV (Nunan et al. 1998). This study was carried out to develop a modified method using a template comprised of total nucleic acid for simultaneous detection of WSSV and TSV in a single tube, 1-step multiplex RT-PCR.
MATERIALS AND METHODS
Experimental infections.Penaeus vannamei, weighing approximately 3.5 g and originating from a commercial shrimp hatchery in Tungkang, southern Taiwan, were reared from post larvae to the juvenile stage in an indoor recirculation system. Randomly selected specimens were checked using TSV RT-PCR (Nunan et al. 1998) and then WSSV PCR (Lo et al. 1996), and all were found to be PCR negative. Juveniles were stocked at a density of about 20 shrimp per 90 l aquarium tank. Inocula were prepared from patently infected (carapace with white spots) P. monodon for WSSV and from P. vannamei for TSV. Shrimp collected from cultivation ponds experiencing disease outbreaks in I-lan, north Taiwan, were proven to be virally infected by WSSV PCR ...
