Laboratory Diagnosis Of Lamivudineassociated Gene Mutations

Methods for the identification of mutations in the HBV P gene include DNA sequencing, restriction fragment length polymorphism analysis, and reverse hybridization. Conventional direct DNA sequencing has been the gold standard method. Both conventional direct DNA sequencing and restriction fragment length polymorphism analysis, however, are labor-intensive and time-consuming methods and thus not useful for the routine diagnostic laboratory. Presently, two assays based on the reverse hybridization principle on DNA sequencing are commercially available.

The INNO-LiPA HBV DR assay (Innogenetics, Ghent, Belgium) is based on the reverse hybridization principle. After extraction, HBV DNA is amplified with a two-round PCR. Both PCR products are visualized on an agarose gel. The second-round PCR product is used for hybridization to the LiPA strip. The probes on the INNO-LiPA HBV DR strip cover codon 180 (wild-type L and mutant M), codon 204 (wild-type M and mutants V and I), and codon 207 (wild-types V, L, and M and mutant I). This assay cannot detect any mutations other than those on the strip but it is highly sensitive and easily applicable.1-17-1

The Trugene™ HBV Genotyping Kit (Bayer HealthCare, Toronto, Ontario, Canada) is a standardized and largely automated sequencing assay. After HBV DNA extraction, a 1.2-kb sequence of the HBV P gene, representing the central portion of the rt domain, is amplified by PCR, and sequencing reactions are then performed on this amplification product with the CLIPTM sequencing (Bayer HealthCare) technology. CLIP sequencing allows both directions of the amplification products to be sequenced simultaneously in the same tube with use of two different dye-labeled primers for each of the four sequencing reactions. Electrophoresis and subsequent data analysis are performed automatically with the automated OpenGene™ and GeneObjects™ DNA sequence analysis system (Bayer HealthCare). Data are acquired with the GeneLibrarian module of GeneObjects software by combination of the forward and reverse sequences. The query sequence is compared with the consensus sequences of HBV genotypes A to G in the Trugene HBV Module of the OpenGene software to determine the HBV genotype of the sample. Mutations in the rt region of the HBV P gene as well as in the overlapping S gene are also automatically detected and reported. The detection limit of this assay is ~ 2.0 x 103 HBV DNA copies/mL, and all viral variants present at concentrations > 20% of the total can be detected. This assay is able to provide HBV genotype and resistance information from the same data and is useful for the molecular diagnostic laboratory.[13]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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