Simultaneous detection of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) by multiplex reverse transcription-polymerase chain reaction (RT-PCR) in pacific white shrimp Penaeus vannamei.
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.
Tsai
Jyh-Ming
JM
King Car Biotechnology Industrial Co, Ltd, Aquaculture Research and Development Center, I-Lan, Taiwan, ROC. anguilla@cm1.hinet.net
Shiau
Lin-John
LJ
Lee
Hsien-Hsiung
HH
Chan
Peter W Y
PW
Lin
Ching-Yu
CY
D000818:Animals; D017756:Aquaculture; D017931:DNA Primers; D004267:DNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D008970:Molecular Weight; D033561:Penaeidae / Q000821:virology*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012335:RNA, Ribosomal / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*
D017931:DNA Primers; D012335:RNA, Ribosomal