La. Jaykus et al., A VIRION CONCENTRATION METHOD FOR DETECTION OF HUMAN ENTERIC VIRUSES IN OYSTERS BY PCR AND OLIGOPROBE HYBRIDIZATION, Applied and environmental microbiology, 62(6), 1996, pp. 2074-2080
This article reports the development of a method to purify and concent
rate intact virions from oyster extracts to a volume and quality compa
tible with viral genomic nucleic acid amplification by reverse transcr
iptase PCR (RT-PCR) and confirmation by oligonucleotide probe hybridiz
ation, Fifty-gram oyster samples were processed by an adsorption-eluti
on-precipitation method and then seeded with 10(1) to 10(5) PFU of pol
iovirus type 1 (PV1) or hepatitis A virus (HAV). Seeded viruses in oys
ter extracts were purified by fluorocarbon extraction and concentrated
by polyethylene glycol (PEG) precipitation and elution, Virus recover
y after elution of PEG precipitates was dependent upon PEG concentrati
on and averaged 60% for PV1 and 40% for HAV, The next processing step
used the protein-precipitating agent Pro-Cipitate (Affinity Technology
, Inc., Brunswick, N.J.) in an adsorption-elution-precipitation scheme
to further concentrate viruses and reduce sample volumes to 100 mu l.
Oyster extracts processed by Pro-Cipitate adsorption-elution-precipit
ation were directly compatible with RT-PCR and yielded virus recoverie
s of > 80% for both PV1 and HAV, When extracts from 50-g oyster sample
s were seeded and processed by the combined concentration and purifica
tion scheme, direct RT-PCR detection of viral genomic RNA was possible
at initial inoculum levels of 10 PFU for both PV1 and HAV and with lo
w levels of Norwalk virus. Virus recoveries based on cell culture infe
ctivity were 25 to 35% for PV1 and 5 to 10% for HAV. When tested on ar
tificially contaminated raw oysters, the combined method successfully
detected greater than or equal to 10(3) PFU of PV1 and HAV and 10(5) R
T-PCR-amplifiable units of Norwalk virus, Virus detection by RT-PCR an
d cell culture infectivity was consistent and well correlated among re
plicate samples and at different virus titers, The procedure developed
in this study is rapid, sensitive, and effective for the direct detec
tion of enteric viruses in oysters by RT-PCR.