A multiplex RT-PCR assay for simultaneous detection of four viruses of shrimp

Viral infection is one of the major causes for the huge economic losses in shrimp farming.A multiplex reverse transcription polymerase chain reaction(mRT-PCR)was developed for simultaneous detection of 4 major shrimp viruses including infectious hypodermal and hematopoietic necrosis virus(IHHNV),Taura syndrome virus(TSV),white spot syndrome virus(WSSV)and yellow-head virus(YHV)in this research.Four sets of specifically designed oligonucleotide primers were used in the assay and each of them could amplify viral nucleic acids by PCR products with different sizes,such as:508 bp for TSV,435 bp for WSSV,301 bp for IHHNV and 614 bp for YHV.Specificity of multiplex RT-PCR nucleic acids of individual virus amplified in PCR reaction containing 4 primer sets were performed and they were highly specific and no specific bands were amplified from other penaeid shrimp pathogenic bacteria.Multiplex RT-PCR for detection of two,three or four types of different viruses in a single reaction system,the corresponding DNA samples of IHHNV(I)and WSSV(W)and cDNA of TSV(T)and YHV(Y)were randomly mixed and amplified using four primer sets.The sensitivity of the multiplex RT-PCR was 0.1 pg for IHHNV,1 pg for TSV,0.02 pg for WSSV and 0.2 pg for YHV.In the field application,49 samples were examined by multiplex RT-PCR.The results were consistent with those results of the single PCR detection.It indicated that this multi-PCR method is superior in terms of sensitivity,specificity rapidity and simplicity,and is potentially a valuable diagnostic tool for shrimp viral infections.