Detection of yellow-head virus (YHV) of Penaeus monodon by RT-PCR amplification

Abstract

A nucleic acid probe was developed using cDNA prepared from ssRNA extracted from yellow-head virus (YHV), a serious pathogen of the black tiger prawn Penaeus monodon. The specificity and sensitivity of this probe was established using dot-blot hybridization with nucleic acid extracts from YHV and from shrimp, bacteria and other viruses. Based on the sequence of this cloned YHV cDNA fragment, a YHV specific primer set for reverse transcription polymerase chain reaction (RT-PCR) of a 135 base pair (bp) sub-fragment was designed for detection of YHV infections in penaeid shrimp. When applied in RT-PCR with templates derived from experimentally or naturally YHV-infected shrimp and with purified YHV or YHV nucleic acid, the expected 135 bp amplification product was obtained. By contrast, nucleic acids extracted from tissue samples of healthy shrimp and from other shrimp pathogens gave no such fragment. This confirmed the specificity of the designed YHV RNA specific primers. RT-PCR based detection demonstrated high sensitivity, in that it could detect 0.01 pg of purified YHV-RNA. In a time course study of an experimental YHV infection, the RT-PCR detection showed evidence of infection at 6 to 12 h post exposure to the virus. However, histopathology typical of YHV infection [i.e. karyorhexis and pycnosis of haemocytes in haematoxylin and eosin (H and E) stained haemolymph smears] was not visible until 42 to 48 h post exposure. The results suggested that RT-PCR might be useful to shrimp aquaculturists for early detection of YHV outbreaks or for detection of asymptomatic carriers.