Nested PCR improves detection of infectious hematopoietic necrosis virus in cells coinfected with infectious pancreatic necrosis virus.
A nested assay using the reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of infectious hematopoietic necrosis virus (IHNV) in cell cultures coinfected with infectious pancreatic necrosis virus (IPNV). Two pairs of primers were designed: one for the amplification of glycoprotein G-specific gene RNA from IHNV (or 1512 bp fragment), and the other for the amplification of an inner 753 bp fragment using the cDNA from the G gene as substrate. Direct RT-PCR was also developed for the amplification of a VP-2 gene fragment from IPNV (613 bp fragment); this method always detected the virus IPNV in the coinfected cells tested but the amplification of IHNV was not as readily achieved. IHNV, however, was detected specifically by nested PCR in coinfected cells at a multiplicity of infection that was 1000 times lower than that of IPNV. Nested PCR was therefore more sensitive than direct RT-PCR for IHNV, and may thus be more appropriate for the detection of low infective titers of IHNV in the presence of IPNV when interference occurs.
Alonso
M
M
Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
Rodríguez
S
S
Pérez Prieto
S I
SI
D000818:Animals; D002460:Cell Line; D018061:Infectious pancreatic necrosis virus / Q000235:genetics / Q000302:isolation & purification*; D010473:Perciformes; D016133:Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012209:Rhabdoviridae / Q000235:genetics / Q000302:isolation & purification*; D012474:Salmon; D012680:Sensitivity and Specificity; D012692:Serial Passage