Je. Barlough et al., NESTED POLYMERASE CHAIN-REACTION FOR DETECTION OF ENTEROCYTOZOON SALMONIS GENOMIC DNA IN CHINOOK SALMON ONCORHYNCHUS-TSHAWYTSCHA, Diseases of aquatic organisms, 23(1), 1995, pp. 17-23
A nested polymerase chain reaction (PCR) was developed for detection o
f the microsporidian parasite Enterocytozoon salmonis in biological sa
mples (blood buffy-coat cells, feces, tissues, lymphocyte cultures) of
chinook salmon Oncorhynchus tshawytscha. A major second-round PCR pro
duct of 407 bp was readily identifiable in ethidium bromide-stained ag
arose minigels. An internal probe was used to verify the identity of t
he amplified product by non-radioactive (digoxigenin-based) Southern b
lotting; final confirmation was made by DNA sequence analysis. A dilut
ion study using infected lymphocytes from in vitro cultures indicated
that a single sound of PCR (35 cycles) was able to detect E. salmonis
DNA from approximately 1000 infected cells. Sensitivity was increased
with the full nested PCR (35 additional cycles), which detected parasi
te DNA from less than or equal to 10 infected lymphocytes. The specifi
city of the PCR was assessed with a panel of microsporidian and myxosp
orean DNAs. In an experimental infection study, E. salmonis DNA was de
tected in blood, feces, and tissues of infected chinook salmon but not
in uninfected control fish.