Dilutional analysis with a nested PCR--a method for quantitating feline immunodeficiency virus provirus in cat tissues.

Important note: Information in this article was accurate in 1994. The state of the art may have changed since the publication date.

Dilutional analysis with a nested PCR--a method for quantitating feline immunodeficiency virus provirus in cat tissues.

Abstr Gen Meet Am Soc Microbiol. 1994;94:485 (abstract no. T-19). Unique Identifier : AIDSLINE ASM94/94313094
Read R; Nie LC; Lu YS; Richardson JA; Pakes SP; University of Texas Southwestern Medical Center, Dallas.

Abstract: 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.

Keywords: 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 ABSTRACT
941030
M94A0860

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