Isolation of HIV-1 RNA from plasma: evaluation of eight different extraction methods.
The efficacy of eight different methods for the extraction of HIV-1 RNA from plasma was compared. The RNA preparation method that gave the best results by RT-PCR was the one described by Chomczynski and Sacchi (1987, Anal. Biochem. 162, 156-159). This method consists of a guanidine thiocyanate treatment followed by three phenol-chloroform-isoamylalcohol extractions and an ethanol precipitation. The disadvantage of this method is that it is time consuming and less suitable for the extraction of large series of samples. Moreover, due to the large number of procedural steps, there is a greater risk of sample mix-up or contamination. Of the single-step RNA purification methods, good results were obtained with the TRIzol method (Gibco Life Technologies, Paisley, UK) and with the extraction method offered by the NASBA kit (Organon Teknika, Turnhout, Belgium). The above single-step methods are recommended since both are sensitive enough to detect low copy numbers of HIV-RNA in the plasma of asymptomatic patients, and require only 2 h for completion. For most of the methods evaluated the inter-test variability was acceptable (mean variation coefficient between duplicate extraction varied between 17.3 and 47.3%). Inter-laboratory reproducibility was evaluated only for the TRIzol-method and found to be low (mean variation coefficient 63.4).
Verhofstede
C
C
Vakgroep voor Klinische Biologie, Microbiologie en Immunologie, Universiteit Gent, Ghent, Belgium.
Fransen
K
K
Marissens
D
D
Verhelst
R
R
van der Groen
G
G
Lauwers
S
S
Zissis
G
G
Plum
J
J
D005069:Evaluation Studies as Topic; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000097:blood* / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*; D014766:Viremia / Q000821:virology
D012367:RNA, Viral