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M9480851.TXT
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1994-09-05
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Document 0851
DOCN M9480851
TI Dilutional analysis with a nested PCR--a method for quantitating feline
immunodeficiency virus provirus in cat tissues.
DT 9410
AU Read R; Nie LC; Lu YS; Richardson JA; Pakes SP; University of Texas
Southwestern Medical Center, Dallas.
SO Abstr Gen Meet Am Soc Microbiol. 1994;94:485 (abstract no. T-19). Unique
Identifier : AIDSLINE ASM94/94313094
AB Most quantitative methods in PCR are subject to error due to the
efficiency with which chosen primers amplify target sequences compared
to reference sequences, and in some systems, the degree to which the
amplified product hybridizes with a labelled probe. To quantitate
provirus in biological samples, we use a highly sensitive nested PCR
which produces the same amount of final amplified product from a single
copy of target sequence as it does with several logs of target sequence.
Samples are diluted until they give positive and negative results and
the most probable concentration of provirus is derived from the Poisson
distribution and the dilution factor. With plasmid DNA as template,
diluted to approximately one copy per sample volume (10 microliters),
the PCR gives results consistent with the expected Poisson distribution.
Biological samples are digested to give crude lysates which show
generally the same threshold effect, although a small percentage of
weaker bands is seen with biological samples. Five cats infected for
three years with the Petaluma isolate of FIV have 6 x 10(2) to 2 x 10(5)
copies of provirus per mm3 lymph node tissue with significant levels in
spleen, liver, lung, salivary gland, gut and bone marrow. The test is
very reliable, non-radioactive and more sensitive than single-stage PCR
but requires multiple determinations to give precise quantitation.
DE Animal Cats DNA, Viral/ANALYSIS Feline Acquired Immunodeficiency
Syndrome/DIAGNOSIS/MICROBIOLOGY Immunodeficiency Virus,
Feline/*ISOLATION & PURIF Organ Specificity Plasmids Poisson
Distribution Polymerase Chain Reaction/*METHODS Proviruses/*ISOLATION
& PURIF Sensitivity and Specificity MEETING ABSTRACT
SOURCE: National Library of Medicine. NOTICE: This material may be
protected by Copyright Law (Title 17, U.S.Code).