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  • ZJM 999094262 So 1

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    Biruk
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    The document describes primers used to detect common mutations associated with resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) via polymerase chain reaction (PCR). It provides the primer sequences, HIV-1 genome location, and expected product length for detecting the M184V, T215Y/F, K103N and Y181C mutations. A table also shows the prevalence of these mutations between two HIV-1 subtypes, determined using both sequencing and allele-specific PCR methods.

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    The document describes primers used to detect common mutations associated with resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) via polymerase chain reaction (PCR). It provides the primer sequences, HIV-1 genome location, and expected product length for detecting the M184V, T215Y/F, K103N and Y181C mutations. A table also shows the prevalence of these mutations between two HIV-1 subtypes, determined using both sequencing and allele-specific PCR methods.

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    © All Rights Reserved
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    4 views4 pages

    ZJM 999094262 So 1

    Uploaded by

    Biruk
    The document describes primers used to detect common mutations associated with resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) via polymerase chain reaction (PCR). It provides the primer sequences, HIV-1 genome location, and expected product length for detecting the M184V, T215Y/F, K103N and Y181C mutations. A table also shows the prevalence of these mutations between two HIV-1 subtypes, determined using both sequencing and allele-specific PCR methods.

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    Table S1: Primers used to detect M184V, T215Y/F, K103N and Y181C mutations.

    Mutations Primer sequence HXB2 location Product length

    NRTIs M184V Wt 5'-TAAATCAGATCCTACATACAAATCATACAT-3' 3099-3128

    Mut 5'-TAAGTCAGATCCTACATATAAATCATCCTC-3' 3099-3128 328bp

    T215Y/F Wt 5'-TTCTTTCTGATGTTTCTTATCTGGTGTGGT-3' 3192-3221

    Mut 5-'TTCTTTCTGATGTTTCTTATCTGGTGGGWA-3' 3192-3221 421bp

    Common forward 5-'CTCAAGACTTCTGGGAGGTCT-3' 2800-2820

    NNRTIs K103N Wt 5'-TCTCCCACATCCAGTACTGTTACTGATGTT-3' 2858-2887

    Mut 5'-TCTCCCACATCCAGTACTGTTACTGAGTTR-3' 2858-2887 300bp

    Y181C Wt 5'-ATCCTACATATAAATCATCCACATATTGRT-3' 3091-3120

    Mut 5'-ATCCTACATATAAATCATCCACATATGGRC-3' 3091-3120 533bp

    Common forward 5'-AGCCAGGAATGGATGGCCCAA-3' 2587-2607

    NRTIs, Nucleoside Reverse Transcriptase Inhibitors; NNRTIs, Non-NRTIs; HXB2, HIV-1 subtype B reference sequence.
    Table S2: HIV-1 drug resistance mutations by HIV-1 subtypes (CRF02_AG vs non-CRF02_AG)

    ARV Class Mutation CRF02_AG (n=53) Non-CRF02_AG (n=22)

    Sequencing (%) ARMS-PCR (%) Sequencing (%) ARMS-PCR (%)

    NRTI M184V 23 (43.3) 27 (50.9) 9 (40.9) 8 (36.3)

    T215Y/F 9 (16.9) 12 (22.6) 5 (22.7) 3 (13.6)

    NNRTI K103N 22 (41.5) 20 (37.7) 1 (4.5) 1 (4.5)

    Y181C 4 (7.5) 6 (11.3) 3 (13.6) 3 (13.6)

    ARV, Antiretroviral; ART, Antiretroviral Treatment; NRTI, Nucleoside Reverse Transcriptase Inhibitor; NNRTI, Non-NRTI. Percentages

    were calculated in each column based on the number of individuals in each group (CRF02_AG or Non-CRF02_AG).
    ARMS-PCR

    primers

    K103N Y181C T215Y M184V

    M WT MUT WT MUT WT MUT WT MUT N Ctrl

    Figure S1: Determination of mutation profile of NIH clone (ARP#12229) with the ARMS-PCR assay. This clone is mutant for amino acid
    (AA) 103 (lane3; 300bp), wild type for AA 181 (lane 4; 533bp), and mutant for AA 215 (lane 9; 421bp), mutant for AA 184 (lane 7; 328bp).
    M, low range DNA ladder (Thermo Scientific); N Ctrl, negative control.
    45 42.6 41.3
    Percentage (%)
    40
    35 30.6
    30 26.6
    25
    18.6
    20 16
    13.3
    15 9.3
    10
    5
    0
    M184V T215YF K103N Y181C
    NRTIs NNRTIs
    Mutations

    Sequencing ARMS-PCR

    a) Patient level

    60
    Percentage (%)
    48.0
    50 42.6
    40
    30.6 29.3
    30
    18.6 20.0
    20 13.3 12.0
    10
    0
    M184V T215YF K103N Y181C
    NRTIs NNRTIs
    Mutations

    Sequencing ARMS-PCR

    b) Population level

    Figure S2: The prevalence of the different mutations as determined by sequence analysis and

    ARMS-PCR when comparing corresponding samples for each patient (a) and at the population level

    (b). NRTI, Nucleoside Reverse Transcriptase Inhibitor; NNRTI, Non-NRTI; HIV-1 drug resistance

    mutations analyzed: M184V, T215Y/F, K103N, Y181C.

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