Evaluation Of HIV-1 Genotype Drug Resistance Assay And Preparation And Evaluation Of Competence Test

Part One Evaluation of three methods for HIV-1drug resistance genotyping testObjectiveTo evaluate the concordance in predicting genotypic drug resistance between in-house method and TRUGENETM or ViroSeqTM method.Methods1.25international proficiency testing (PT) samples received from2009to2013were detected by three menthods, then pair-wise comparison results was analyzed to validate their concordance on drug resistance mutation and drug resistance report.2. To further confirm the results,15serum specimens were detected by in-house and TRUGENETM methods, then compared their results concordance.Results1. Resistance-associated mutations consistent evaluation:for25PT samples,99.42%(2933/2950) drug resistance mutation sites can be detected by all three methods, all pairwise comparison Kappa values>0.81(P<0.01).2. Drug resistance test results consistent evaluation:inconsistent comparison results are mainly concentrated in the four nucleoside reverse transcriptase inhibitors zidovudine (AZT), didanosine (ddl), stavudine (d4T), abacavir (ABC), and mainly for minor inconsistencies. Where in-house with ViroSeqTM minor inconsistencies were28%(7/25),28%(7/25),16%(4/25),20%(5/250, in-house with TRUGENETM were44%(11/25),28%(7/25),36%(9/25),28%(7/25); TRUGENETM with ViroSeqTM were24%(6/25),8%(2/25),28%(7/25).8%(2/25). When the In-house was comparied with ViroSeqTM, the drug AZT. in-house was comparied with TRUGENETM, drugs ddl, d4T, ABC, tenofovir (TDF) were moderate agreement (weighted Kappa coefficients were0.54,0.44,0.52,0.42,0.59, P<0.01), the rest of the comparison results were highly consistent (weighted Kappa coefficient was0.61to0.80) or the consistency of strong (weighted Kappa coefficient0.80).3. For15serum specimens,99.55%(1762/1770) drug resistance mutation sites can be detected by the two methods, the difference about drug results concentrated in AZT\ddI、d4T、ABC.ConclusionThe in-house genotyping system had a high concordance with commercial TRUGENETM or ViroSeqTM genotyping system. Part Two Preparation and evaluation of HIV-1drug resistance proficiency testing materialObjectiveTo prepare HIV-1drug resistance proficiency testing material and evaluate its homogeneity and stability.Methods1. Extracting HIV-1viral nucleic acids from positive plasma, then obtaining3100bp target gene by RT-PCR and second round of PCR2. The target gene was connected with pMDTM19-T vector, the recombinant vector was introduced into JM109competent cells, and inoculated LB agar medium coated with Amp (ampicillin) and X-Gal,37℃overnight. White colonies were picked from the medium and were inoculated in LB broth, overnight shaking culture, PCR identification, extracting recombinant plasmid from positive bacteria and sequencing.3. Searching data to determine real-time PCR primers and the reaction system, determining the annealing temperature by gradient annealing real-time PCR.4. Diluting recombinant plasmids by negative plasma, determining the optimum concentration by real-time PCR Ct value. Diluting, packaging and preparing testing materials.5. Choosing three different recombinant plasmids of different concentrations, each drawing10, numbering, detecting Ct value by real-time PCR methods. Taking200ul from each sample, mixing well. Mixed samples for detection of intra-assay imprecision, each sample detected10times. Making homogeneity of variance test by the two sets of data (P>0.05was considered no significant difference and the product had a well uniformity), testing the samples’uniformity.6. The three samples of different concentrations incubator at37℃, room temperature (20-25℃, relative humidity20-25%),4℃was placed1,3,5,7,10,15,20days, and -20℃,-80℃repeated freezing and thawing to room temperature5times, detecting the real-time PCR Ct value range and determining the stability of the sample.Results1.5target genes were acquired, there were01(B subtype, drug sensitive),02(B subtype drug resistance),03(B subtype, drug resistance),04(BC subtype, drug resistance),05(AE subtype, drug resistance).2. Connecting target genes and vectors to obtain recombinant vectors V1, V2, V3,V4, V5, the sequencing results of the PCR product was identified by homology, the step value is100, the process of gene cloning had good stability.3. Gradient annealing time PCR results showed that when the annealing temperature is64.1℃, Ct value of each sample was minimum, so the real-time PCR annealing temperature is determined at64.1℃.4. Sample diluting experimental results showed that when the plasmids were diluted less than10000-fold, the real time PCR Ct values were less than30. So, diluting every plasmid10000-fold making testing materials.5. Selecting three different sample V1、V2、V3, do uniformity test. Ct values of the two groups of homogeneity of variance test results showed that:the before and after mean of three samples were similar, the display does not have a statistically significant difference (P>0.05). Therefore, we can consider the testing materials made by recombinant plasmid diluted uniformity is good.6. Selecting V1,V2, V3do stability testing, the results showed:the testing materials were stable for at least at room temperature and4℃20days (Ct values did not change significantly),37℃after3days concentrations lower in varying degrees (Ct value becomes large); stored at-20℃and-80℃repeatedly frozen five times concentrations were not changed significantly (Ct values did not change significantly). ConclusionIn this study, in-house HIV-1drug resistance proficiency testing materials prepared by cloning vectors were verified stability and uniformity well, which solved the problem of large quantities of drug samples is difficult to achieve. This was an economical and convenient method of preparing in-house HIV-1genotypic resistance proficiency testing materials.