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).