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Journal of Clinical Microbiology, November 2003, p. 4971-4979, Vol. 41, No. 11
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.11.4971-4979.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Novel Enzyme-Linked Minisequence Assay for Genotypic Analysis of Human Immunodeficiency Virus Type 1 Drug Resistance

Wataru Sugiura,1* Kazunori Shimada,2 Masakazu Matsuda,1 Tomoko Chiba,1 Lay Myint,1 Aiko Okano,1 and Kaneo Yamada3

AIDS Research Center, National Institute of Infectious Diseases, Tokyo 2080011,1 Genome Science Laboratories Co., Ltd., Matsukawa, Fukushima 960-1242,2 Japanese Foundation for AIDS Prevention, Minatoku, Tokyo 105-0001, Japan3

Received 8 January 2003/ Returned for modification 23 April 2003/ Accepted 12 August 2003

We constructed a novel tool for genotypic analysis of human immunodeficiency virus type 1 (HIV-1) drug resistance by using an enzyme-linked minisequence assay (ELMA). ELMA is a combination of hybridization and a 1-base extension reaction, and we designed the assay to detect five mutations conferring nucleoside analogue resistance (M41L, D67N, K70R, T215Y, and M184V) and six mutations conferring protease inhibitor resistance (D30N, M46I, G48V, V82A, I84V, and L90M). At all detection points, ELMA demonstrated high sensitivity and specificity, sufficient for clinical use. Compared to that obtained by direct sequencing, the genotypic information obtained by ELMA is limited to the targeted loci for which it was designed. However, ELMA proves advantageous in several respects. The assay does not require expensive equipment, such as an autosequencer, and can be performed in regular clinical diagnostic laboratories. Therefore ELMA can be a candidate for a drug resistance monitoring assay to be introduced in developing countries. In addition, ELMA demonstrated higher sensitivity in the detection of minor resistant populations. We successfully detected a minor virus population (10%) by the assay. The high sensitivity and specificity of the assay recommend it as a first screening assay for drug resistance surveillance.

* Corresponding author. Mailing address: AIDS Research Center, National Institute of Infectious Diseases, 4-7-1 Gakuenn Musashimurayama, Tokyo 2080011, Japan. Phone: 81-42-561-0771. Fax: 81-42-561-7746. E-mail: wsugiura{at}nih.go.jp.

Journal of Clinical Microbiology, November 2003, p. 4971-4979, Vol. 41, No. 11
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.11.4971-4979.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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