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Antimicrobial Agents and Chemotherapy, December 2002, p. 3861-3868, Vol. 46, No. 12
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.12.3861-3868.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mutagenically Separated PCR Assay for Rapid Detection of M41L and K70R Zidovudine Resistance Mutations in CRF01_AE (Subtype E) Human Immunodeficiency Virus Type 1

Lay Myint,¹ Koya Ariyoshi,¹ Hua Yan,¹ Alexander J. Frater,² Wattana Auwanit,³ Panita Pathipvanith,⁴ Kaneo Yamada,⁵ Masakazu Matsuda,¹ Tomoko Chiba,¹ Kazunori Fujita,¹ Myra McClure,² Jonathan N. Weber,² and Wataru Sugiura^1*

AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan,¹ Wright Fleming Institute, Faculty of Medicine, St. Mary's Campus, Imperial College, London, United Kingdom,² National Institute of Health, Department of Medical Science, Thailand,³ Lampang Hospital, Lampang, Thailand,⁴ Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Japan⁵

Received 25 April 2002/ Returned for modification 19 July 2002/ Accepted 12 September 2002

A rapid zidovudine (ZDV) resistance genotypic assay was developed based on the mutagenically separated PCR (MS-PCR) technique to detect two ZDV-resistant mutations, M41L and K70R in CRF01_AE (subtype E). Endpoint dilution analysis revealed that the newly constructed MS-PCR assay could successfully detect three to nine copies of human immunodeficiency virus type 1 template RNA. The test against wild-type and mutant template mixtures in different ratios demonstrated that the assay could detect 10% minor population, at least. Fifty-one subtype E clinical samples were analyzed by the newly constructed MS-PCR assay and direct nucleotide sequencing. The concordance of the two assays was 92 and 100% in codons 41 and 70, respectively. The MS-PCR assay is a rapid, simple, and inexpensive assay that is highly sensitive in detecting mutant targets, including minor populations. Thus, it could be used as a powerful tool for epidemiological surveillance of drug-resistant mutations in developing countries.

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* Corresponding author. Mailing address: AIDS Research Center, National Institute of Infectious Diseases, 4-7-1 Gakuenn, Musashimurayama, Tokyo 208 0011, Japan. Phone: 81-42-561-0771. Fax: 81-42-561-7746. E-mail: wsugiura{at}nih.go.jp.

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Antimicrobial Agents and Chemotherapy, December 2002, p. 3861-3868, Vol. 46, No. 12
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.12.3861-3868.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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