Fully automated quantification of human immunodeficiency virus (HIV) type 1 RNA in human plasma by the COBAS AmpliPrep/COBAS TaqMan system.

BACKGROUND: HIV-1 RNA is a key parameter for reliable diagnosis and treatment of HIV-1 infection. The determination of HIV-1 RNA reduces the pre-seroconversion period in the diagnosis of HIV-1 infection and supports clinical management of HIV-1-infected patients. OBJECTIVES AND STUDY DESIGN: The COBAS AmpliPrep/COBAS TaqMan HIV-1 Test combines automated extraction of total nucleic acids on the COBAS AmpliPrep Instrument with real-time PCR on the COBAS TaqMan Analyzer, thus greatly reducing hands-on time during sample preparation and amplification/detection. The test was evaluated for sensitivity, dynamic range, precision, subtype inclusivity, interfering substances, diagnostic and analytical specificity, as well as correlation with three other commercial tests for HIV-1 RNA quantification. RESULTS: The COBAS AmpliPrep/COBAS TaqMan HIV-1 Test demonstrated an assay sensitivity of 40 copies/mL, a greater than 5 log(10) measuring range of 40-1.0E+07 copies/mL (1.6-7.0 log(10)) and a reliable determination of HIV-1 group M and N subtypes in EDTA plasma. Quantification results were highly correlated with those obtained by the COBAS AMPLICOR HIV-1 MONITOR Test v1.5, the COBAS AmpliPrep/COBAS AMPLICOR HIV-1 MONITOR Test v1.5 and the VERSANT HIV-1 RNA 3.0 assay. CONCLUSIONS: The COBAS AmpliPrep/COBAS TaqMan HIV-1 Test excellently satisfies the requirements for reliable quantification of HIV-1 RNA in clinical specimens by a broad linear measuring range and a fully automated quantification procedure. It is highly appropriate for therapy monitoring and routine management of HIV-1 infection.

Characterization of the corrinoid iron-sulfur protein tetrachloroethene reductive dehalogenase of Dehalobacter restrictus.

The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 +/- 1 kDa, whereas the native molecular mass was 71 +/- 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-beta-D-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0 +/- 0.1 mol of cobalamin, 0.6 +/- 0.02 mol of cobalt, 7.1 +/- 0.6 mol of iron, and 5.8 +/- 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 +/- 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The K(m) values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4 +/- 3.2, 23.7 +/- 5.2, and 47 +/- 10 micro M, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced. Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences. Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy.