PROCAARE: CLINICAL SCIENCE--HIV RNA QUANTITATION METHODS

[Albert Shaw <ashaw@usa.healthnet.org>]

KEYWORDS: RNA/ POLYMERASE CHAIN REACTION/ SEQUENCE-BASED AMPLIFICATION/ 
BRANCHED DNA
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Reference: Caliendo, A.M.  (1995). Laboratory methods for quantitating 
HIV RNA.  AIDS Clin. Care 7: 89-96.

With recent data establishing the superiority of plasma HIV RNA in 
providing prognostic information and in monitoring response to therapy, 
three methods for quantitation of RNA have become available. 

I. Polymerase chain reaction (PCR)--Amplicor HIV Monitor Test (Roche 
Molecular Systems)

	Plasma samples may be collected in EDTA or acid citrate dextrose 
(not heparin); however, it should be noted that samples in acid citrate 
dextrose may give 20% lower results because of a larger volume of 
anticoagulant.  200 microliters of plasma are treated with the chaotropic 
agent guanidinium isothiocyanate to lyse viral particles (rendering the 
samples noninfectious) and release RNA, which is then precipitated with 
isopropanol.  Reverse transcriptase is then used to convert the 
single-stranded RNA to complementary DNA (cDNA).
	The cDNA specimens are subjected to successive rounds of PCR, 
which consist of heat denaturation to separate the DNA strands, annealing 
of HIV-specific primers, and replication using a thermostable DNA 
polymerase; as a result, the cDNA can be readily amplified by six orders 
of magnitude.  Quantitation of amplified products is achieved through the 
use of an internal RNA standard, which is recognized by the same PCR 
primers but can be distinguished by sequence rearrangement from HIV RNA.  
The standard is included in the reaction mixture during sample 
preparation and amplification, and by adding a known number of copies of 
the standard, the copy number of the sample may be determined.  The 
dynamic range of this PCR-based method is three orders of magnitude; the 
lower range of detection is 500 copies/ml.

II. Nucleic acid sequence-based amplification (NASBA; Organon Technika)

	This procedure may be used for plasma, serum, whole blood, CSF, 
semen, or urine.  Plasma samples (100 microliters required) may be 
collected in EDTA, acid citrate dextrose, or heparin.  RNA is extracted 
from the sample using guanidinium isothiocyanate and acidified silica 
(again rendering the sample noninfectious).   In contrast to PCR, RNA is 
directly amplified; an oligonucleotide primer containing a promoter 
sequence for RNA polymerase from the bacteriophage T7 is first annealed 
to the sample.  Reverse transcriptase then generates a double-stranded 
cDNA of the sample.  Finally, T7 RNA polymerase synthesizes large numbers 
of RNA copies from this cDNA (nearly a billion copies can be made in 90 
minutes); the signal is detected using a chemiluminescence assay.  Again, 
inclusion of internal standard RNA is employed to facilitate quantitation 
of the sample.  The limit of detection of NASBA is 400 copies/ml, with a 
working range of detection of four orders of magnitude.

III. Branched DNA amplification (Quantiplex HIV RNA assay; Chiron)

	This method requires one ml. of plasma, which can be collected in 
acid citrate dextrose or EDTA, but not heparin.  Plasma HIV is 
concentrated by centrifugation, and the viral particles are lysed to 
release RNA.  This RNA is bound to a microtiter plate coated with 
HIV-specific probes.  The branched DNA method involves amplification of 
this bound RNA, using first binding of branched DNA molecules recognizing 
the immobilized RNA, then subsequent hybridization of branched 
DNA-specific oligonucleotide molecules which are linked to alkaline 
phosphatase.  The amplified signal is then detected using a 
chemiluminescent assay.  Quantitation of sample RNA is achieved by 
generating a standard curve in parallel with patient assays.  The dynamic 
range of this assay is 10,000 to 1.6 million RNA copies/ml; the initially 
reported lower range of detection of 10,000 RNA copies/ml has apparently 
been improved to nearly 500/ml.

For all of these assays, meticulous technique is essential to prevent 
cross-contamination of patient specimens; for the PCR and NASBA 
techniques, "carryover" contamination of amplified nucleic acids should 
be rigorously excluded.