title: Comparison of 3 nucleic acid isolation methods for the quantification of HIV-1 RNA by Cobas Taqman real-time polymerase chain reaction system.
abstract: Quantification of human immunodeficiency virus type 1 (HIV-1) RNA viral load in blood is a clinically validated tool for the management and follow-up of patients infected with this virus. HIV-1 RNA quantification can be performed by several kinds of commercially available polymerase chain reaction (PCR) assays. The sensitivity of these assays is affected by the nucleic acid isolation procedures that are performed before PCR. Especially for the samples containing low numbers of HIV-1 RNA, an effective nucleic acid isolation method is very important to obtain a high nucleic acid output. The purpose of this study was to compare and evaluate the conventional manual extraction method, automated MagNA Pure system, and automated AmpliPrep system for the nucleic acid isolation before TaqMan real-time PCR assay for the quantification of HIV-1 RNA in plasma samples. Plasma samples of 40 patients in which anti-HIV antibodies were found as positive serologically were included in this study. Nucleic acid isolation from these samples was performed by manual High Pure Viral Nucleic Acid Isolation kit (Roche Diagnostics, Mannheim, Germany), automated MagNA Pure LC Total Nucleic Acid Isolation kit (Roche Diagnostics), and automated Cobas AmpliPrep kit (Roche Diagnostics). The nucleic acids obtained by these 3 methods were amplified and detected in the Cobas Taqman 48 (Roche Diagnostics) instrument. Viral RNA copies detected by AmpliPrep system, MagNA Pure system, and manual isolation method were found to be between 40 and 420, 000 (average, 100 135), 53 and 1 200 000 (average, 220 554), and 183 and 590 000 (average, 104 761) copies/mL, respectively. The results of isolation methods were evaluated for each sample, and it was found out that the number of HIV-1 RNA obtained by MagNA Pure system was higher than the other 2 methods. All of the 3 methods were found to be reliable in detecting very small amounts of HIV-1 RNA in samples. Manual isolation method is a cheaper alternative, but because the nucleic acid isolation can be performed in a shorter time by automated systems when compared with manual isolation method, these systems can be preferred especially in the laboratories that have high number of samples.
authors.affiliation: Department of Microbiology and Clinical Microbiology, Hacettepe University Faculty of Medicine, Ankara, Turkey. aalp@hacettepe.edu.tr
authors.forename: Alpaslan
authors.initials: A
authors.lastname: Alp
authors.forename: Gülsen
authors.initials: G
authors.lastname: Hascelik
mesh_terms: D005858:Germany; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D009696:Nucleic Acids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
chemical_list: D009696:Nucleic Acids; D012367:RNA, Viral