AEGiS-11IAC: Sequencing method for genetic analysis of HIV-1 in Thailand.

11th International AIDS Conference


Vancouver, British Columbia — July 7-12, 1996

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Sequencing method for genetic analysis of HIV-1 in Thailand.

Int Conf AIDS 1996 Jul 7-12; 11:440 (abstract no. Pub.A.1017)
Isarangkura Na Ayuthaya P, Daungchandra S, Saipradit N, Sriwanthana B, Naganawa S, Auwanit W, Bumisawasdi J, Jayavasu C, Miyamura K, Honda M, Yamazaki M; Department of Medical Science, Ministry of Public Health, Thailand. Fax: 662-5899869. E-mail: watimm@health.moph.go.th.

OBJECTIVE: To establish an appropriate sequencing method of genetic analysis for characterization of HIV-1 prevalence in Thailand using autosequencer.

METHODS: Genomic DNA were extracted from PBMCs of 20 HIV-1 infected patients (10 IDUs, 10 heterosexuals), were amplified by nested PCR for HIV V3 domain using a nested PCR techniques. After amplification using two sets of outer primers (MK603/CO602 and OA3/OD) and one set of the inner primer (EB2/EC2), PCR products were purified by Centricon centrifugation .Purified PCR products were direct-sequenced by dye-terminator method using automated DNA sequencing system. In addition, the PCR products were cloned into pUC19 and transformed into E. coli strain XL-1 blue. Clones containing DNA inserts were sequenced by dye-primer method. Nucleotide and amino acid sequence analysis were determined by Sequence Navigator and Genetyx computer software..

RESULTS: Our results showed that the MK603/ CO602 outer primer set was capable of amplifying V3 region of HIV-1 env gene of both clade E and B viruses while the OA3/ OD3 did amplify only some of clade E viruses. We found one of IDUs was infected by both clade E and B by OA3/ OD3 amplification. Sequencing using either dye-terminator or dyeprimer method, however, has revealed the corresponding clades among all samples tested.

CONCLUSION: We established sequencing method for genetic analysis of field viruses in Thailand. Clades of viruses from sequencing data are basically similar as compare to peptide-based ELISA. The direct dye-terminator method is simple and accurate for determination the clade of HIV-1, suggesting that it is an appropriate method for molecular-epidemiology study.

Keywords: AEGIS, HIV-1, Genes, env, Polymerase Chain Reaction, Heterosexuality, Thailand, Human, methods, genetics, ICA11KWDaegis,hiv-1,genes,env,polymerasechainreaction,heterosexuality,thailand,human,methods,genetics,ica11

960707
PubA1017

Copyright © 1996 - International AIDS Society (IAS). Reproduction of this abstract (other than one copy for personal reference) must be cleared through the IAS.