A rapid phenotypic assay for detection of acyclovir-resistant varicella-zoster virus with mutations in the thymidine kinase open reading frame.

ABSTRACT: Susceptibility assays by cell culture methods are time-consuming and are particularly difficult to perform with varicella-zoster virus (VZV). To overcome this limitation, we have adapted a functional test of the viral thymidine kinase (TK) in TK-deficient (tdk mutant) bacteria to detect ACV-resistant VZV in clinical samples. After PCR amplification, the complete viral TK open reading frame (ORF) is purified from PCR primers, digested with two restriction enzymes, and ligated in an oriented fashion into a bacterial expression vector. The ligation products are then used to transform tdk mutant bacteria. After transformation, an aliquot of the bacteria is plated onto a plate with minimal medium containing (i) ampicillin to select for plasmids carrying the viral TK ORF and (ii) isopropyl beta-

SUBMITTER: Sahli R 

PROVIDER: S-EPMC89785 | biostudies-literature | 2000 Apr

REPOSITORIES: biostudies-literature