Molecular Genetic Testing

Phenotypic methods require previous isolation of viral strains on cell cultures, which is time consuming and delays the change of antiviral treatment according to in vitro susceptibility. Genotypic tests are now being developed to detect resistant virus within a shorter time. The viral genes encoding the two targets of antiviral drugs (TK and DNA polymerase) are amplified by PCR and PCR products are then sequenced. The difficulty lies in the fact that numerous nucleotide substitutions may be found and these must then be identified as mutations responsible for resistance or not. This interpretation will be made easier as more and more results on mutations detected in resistant isolates are collected. In addition, site-directed mutagenesis studies are in progress to formally assess the role of the mutations in the development of antiviral drug resistance. Results already obtained by genetic characterization of clinical isolates resistant to ACV indicate that sequencing of limited areas in the TK and DNA polymerase genes is sufficient to detect most of the mutations associated with resistance. For TK, which is only 1128 nucleotides long, it is preferable to sequence all the gene, but sequencing of nucleotides 150 to 600, combined with a specific detection of mutations at codon 336, should allow the detection of 80% of the mutations associated with resistance. For DNA polymerase, we focused on the region between nucleotides 1500 and 3000, which includes 90% of the mutations reported in resistant strains.

At the present time, genetic detection of ACV-resistant HSV is based on PCR amplification and sequencing of

Table 1 Primers for TK and DNA polymerase genetic characterization

Target gene

Primers for PCR

PCR product length and localization

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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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