3rd Conference on Retroviruses and Opportunistic Infections


Washington, DC - January 28-February 1, 1996

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LINE PROBE ASSAY (LIPA) FOR THE RAPID AND RELIABLE DETECTION OF DRUG INDUCED MUTATIONS IN THE HIV RT GENE.

Conf Retroviruses Opportunistic Infect 1996 Jan 28-Feb 1; 3rd:60 (abstract no. 40)

Stuyver L, Wyseur A, Scarcez T, Verhofstede C, Louwagie J, Rossau R
Innogenetics, 9052 Gent, and University Hospital, 9000 Gent, Belgium.

BACKGROUND: HIV infected patients become gradually resistant to nucleoside analogues (AZT, ddI, ddC, 3TC) by the stepwise accumulation of mutations in the reverse transcriptase (RT) gene. A quick and reliable way to screen for mutations is essential.

DESIGN: LiPA is based on the reverse hybridization principle: short oligonucleotide probes are immobilized on membrane strips as parallel lines; biotynylated PCR products are hybridized against those probes under highly stringent conditions; after washing, streptavidin-alkaline phosphatase is bound to any labeled hybrid previously formed; addition of substrate results in a purple brown precipitate.

METHODS: Wild type and mutant probes were designed for the HIV RT gene at clinically important codon positions. A selection of PCR fragments of the RT gene covering the region from aa 28 to aa 220 were cloned and recombinant clones were sequenced in both directions.

RESULTS: This panel of recombinant and highly characterized clones served as a quality control panel to optimize for probes at each of the positions. Only probes that allowed to discriminate between one single nucleotide mismatch were retained. A single assay was able to detect the following drug induced mutations: M41L, T69D, K70R, L74V, M184V, F214L, T215Y, and T215F, as well as polymorphisms for E40, K73, V75, Q182, and G213.

CONCLUSION: The current selection of probes allowed the reliable detection of cloned mutations and polymorphisms. At least 90% of patients sera studied could unambiguously and rapidly be characterized.

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