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A Comparison of Filter Capture and Precultivation for Quantitative Detection of Infectious Hematopoietic Necrosis Virus (IHNV) by using Polymerase Chain Reaction (PCR) Combined with the Most Probable Number (MPN) Method
Author(s) -
Yoshinaka Toko,
Yoshimizu Mamoru,
Ezura Yoshio
Publication year - 1999
Publication title -
journal of aquatic animal health
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 52
eISSN - 1548-8667
pISSN - 0899-7659
DOI - 10.1577/1548-8667(1999)011<0154:acofca>2.0.co;2
Subject(s) - infectious hematopoietic necrosis virus , biology , polymerase chain reaction , virus , virology , real time polymerase chain reaction , reverse transcription polymerase chain reaction , gene , microbiology and biotechnology , gene expression , genetics , fish <actinopterygii> , rainbow trout , fishery
Infectious hematopoietic necrosis virus (IHNV) was collected from phosphate‐buffered solutions and water that contained low numbers of the virus either by precultivation in cell cultures or capture by membrane filtration. The RNA from IHNV present in precultures or on membrane filters was extracted, and a 510‐base‐pair segment of the viral gene coding for the N‐protein was amplified by reverse transcription polymerase chain reaction (RT‐PCR). The number of virus particles present in the original sample was then calculated by using the most probable number (MPN) method. Low numbers of IHNV as preset in the original inoculum were accurately estimated by this PCR–MPN with precultivation. The PCR–MPN was less effective with the filter capture than the precultivation method because the quantity of IHNV detected was only 1/10 of that found in the original inoculum.