| Objective According to the principle of allele specific PCR, a series of primers were designed aiming to detect drug resistant mutations in the reverse transcriptase coding region of the HIV-1 subtype CRF01_AE, which is the popular HIV-1 subtype spreading in Guangxi. To explore the feasibility of using ASPCR method to detect HIV-1 drug resistance mutations and to develop a simple, sensitive, high efficiencyand low cost method for clinical drug resistance testing as well as epidemiological investigation.Method The DNA fragment of HIV-1 CR01_AE pol gene was cloned into pUCm-T vector to obtain wild type plasmid which contains wild-type pol gene. A series of mutant plasmids that contain various drug resistant mutations were then obtained by conducting the site-directed mutations into wild type plasmid, respectively. According to the principle of allele specific PCR, a universal reverse primer was designed for detecting wild type gene and all of mutant genes. For each mutation stie, wild-type primers and mutant-type primers were designed, to detect wild-type site and mutant site respectively.Using various plasmids as templates, the designed primers were tested by PCR individually to determin their specificity and sensitivity. The ASPCR assay was then validated by the clinical specimens from ART-experienced patients and was compared to the results determined by Sanger sequencing.Results1. HIV-1 subtype CRF01_AE wild type plasmid and 19 mutant type plasmids were constructed successfully. Each mutant plasmid carries one drug resistant mutantion in the reverse transcriptase coding region. All cloning sequences were validated by Sanger sequencing.2. For all 19 sites in reverse transcriptase region, a universial downstream primer was designed. For each site, two wild tpye primers and two mutant type primers were designed. The 19 sites include 10 NRTIs resistant mutant sites(M41L、D67N、K65R、K70R、K70E、M184V、M184I、L210W、T215Y、T215F) and 9 NNRTIs resistant mutant sites (K103N、V108I、E138A、V179D、Y181C、Y188C、G190A、G190R、M230I).3. Among all 19 sites, on 15 sites we identified that at least one wild type primer was suitable for detection of each wild type template. Among 15 sites, the same wild type primer can be used for M184V and M184I, G190R and G190A,T215Y and T215F, respectively. On 16 sites, we identified one mutant type primer was suitable for detection of each mutant type template. Among identified primers, 3 primers could detect 1% mutant type template; 9 primers could detect 5% mutant type template; and 4 primers could detect 10% mutant type template.3.From 23 clinical specimens, Sanger sequencing found that 6 samples contain K103N mutation, 9 samples contain Y181C mutation,and 5 samples contain G190A mutation. Corresponding numbers determined by our ASPCR assey were 4, 7 and 5, respectively. The conformity between ASPCR and Sanger sequencing was 66.6% , 77.8%, and 100% in the three mutations respectively.Conclusion This study explores the feasibility of using ASPCR method to detect HIV-1 drug resistant mutations in pol gene region of HIV-1 subtype CRF01_AE Among 19 drug resistance sites, we sucessfully desgined wild type primers and mutant type primers for mutation detection on 15 sites, , which,obviously, has laid a solid foundation for the next step of establishing a microarray-based solid-phase multiple ASPCR chip to detect HIV-1 drug resistant mutations. |
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