Registered Representative Drug Tested Regularly?

doi: 10.1089/aid.2014.0059. Epub 2014 Dec 3.

Japanese external quality cess program to standardize HIV-i drug-resistance testing (JEQS2010 program) using in vitro transcribed RNA as reference cloth

Junko Hattori, Masakazu Matsuda, Kiyomi Okada, Yukumasa Kazuyama, Osamu Hashimoto, Shiro Ibe, Shin-ichi Fujisawa, Hitoshi Chiba, Masashi Tatsumi, Shingo Kato, Wataru Sugiura

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• PMID: 25469535
• PMCID: PMC4347889
• DOI: ten.1089/aid.2014.0059

Gratis PMC commodity

Japanese external quality assessment program to standardize HIV-1 drug-resistance testing (JEQS2010 program) using in vitro transcribed RNA as reference material

Shigeru Yoshida  et al. AIDS Res Hum Retroviruses. 2015 Mar .

Complimentary PMC article

Abstract

To design advisable antiretroviral therapy regimens and avoid the emergence of human immunodeficiency virus (HIV)-1 variants with reduced susceptibility to antiretroviral drugs, genotypic drug-resistance testing (HIV genotyping) is strongly recommended. To monitor the quality of HIV genotyping in Japan, we performed an external quality assessment (EQA), named the Japanese external quality assessment program, to standardize HIV genotyping (JEQS). To accurately evaluate the quality of HIV genotyping, nosotros employed as reference material (RM) a well-characterized sample, in vitro transcribed RNA (trRNA) that includes the HIV gag-political leader sequence, and created a JEQS2010 panel consisting of 3 single variant and three mixed trRNA samples. All 11 participating laboratories showed high cyclopedia rates (>96%) for the single variant samples. Eight laboratories also showed good rates of detecting pocket-sized variants, but 3 laboratories failed to detect the variants comprising half of the sample. These three laboratories used a common primer that had iv internal mismatches to the small trRNA clone. This program showed the usefulness of trRNA every bit RM, the loftier quality of HIV genotyping, and extensive interlaboratory variation in the ability to detect pocket-size variants. These results advise that improving the quality of HIV genotyping in Japan requires regularly implementing the EQA program and improving the HIV genotyping protocol in each laboratory.

Figures

FIG. 1.

Construction of plasmid for HIV-1 gag–politician RNA transcription. (A) An amplified hepatitis C virus (HCV) 5′-UTR fragment (nt 19–339, the accession number of the reference sequence is AB049088) is ligated into the TA cloning site of pGEM-T Easy vector. (B) The amplified HIV gag–pol fragment (nt ane,480–4,315, reference strain is HXB2, K03455) is ligated into pGEM-HCV, followed by HIV gag–politico RNA transcription by T7 RNA polymerase.

FIG. two.

Cyclopedia rates over the entire nucleotide sequence. (A) Samples #14, #fifteen, and #16 consist of a single variant transcribed RNA (trRNA). Concordance rates reflect consummate agreement between reported sequences and reference. (B) Samples #26 and #28 consist of 2 trRNAs containing a minor variant with thirty% and 10% abundance, respectively. Sample #29 contains a variant comprising half of the sample.

FIG. three.

Detection rate of the minor variant. (A) Detection rate of the nucleotide sequence of the minority trRNA clone in sample #26 (thirty% minority). (B) Detection rate of the nucleotide sequence of the minority trRNA clone in sample #28 (10% minority). Both samples have 88 discordant positions; theoretically 26 positions are in protease (PR) and 62 positions are in reverse transcriptase (RT).

FIG. 4.

Sequence comparison betwixt primers and trRNA in a mixed sample. Primer set DRRT1L and DRRT4L is for reverse transcriptase polymerase chain reaction (RT-PCR) and DRRT7L and DRRT6L for nested PCR. Identical bases are represented past dashes (-).

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