PCR assay for discriminating between infectious hypodermal and hematopoietic necrosis virus (IHHNV) and virus-related sequences in the genome of Penaeus monodon.
We developed a PCR assay that can detect infectious hypodermal and hematopoietic necrosis virus (IHHNV) but that does not react with IHHNV-related sequences in the genome of Penaeus monodon from Africa and Australia. IHHNV is a single-stranded DNA virus that has caused severe mortality and stunted growth in penaeid shrimp. Recently, IHHNV-related sequences were found in the genome of some stocks of P. monodon from Africa and Australia. These virus-related sequences have a high degree of similarity (86 and 92% identities in nucleotide sequence) to the viral genome, which has often generated false-positive reactions during PCR screening of these stocks. For this assay, a pair of IHHNV primers (IHHNV309F/R) was selected. The sequences of these primers match (100% of nucleotides) the target sequence in IHHNV, but mismatch 9 or 12 nucleotides of the genomic IHHNV-related sequences. This PCR assay was tested with various IHHNV isolates and with a number of samples of shrimp DNA that contained IHHNV-related sequences. This assay can reliably distinguish IHHNV DNA from shrimp DNA: it only detects IHHNV. Also, this pair of primers was included in a duplex PCR to detect IHHNV and simultaneously determine the presence of an IHHNV-related sequence. Using these primers, the PCR assay has a sensitivity equivalent to a PCR assay commonly used for detecting IHHNV in Litopenaeus vannamei, and can be used for routine detection.
Tang
Kathy F J
KF
Department of Veterinary Science and Microbiology, University of Arizona, Tucson, Arizona 85721, USA. fengjyu@u.arizona.edu
Navarro
Solangel A
SA
Lightner
Donald V
DV
D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics*; D019223:Densovirinae / Q000235:genetics* / Q000472:pathogenicity*; D014644:Genetic Variation; D016678:Genome; D033561:Penaeidae / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013047:Specific Pathogen-Free Organisms
D017931:DNA Primers; D004279:DNA, Viral