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目的制备人类免疫缺陷病毒1型(HIV-1)基因型耐药检测能力验证考核品,并对其均匀性和稳定性进行评估。方法选取前期检测出的5个HIV-1阳性血浆样本,其中4个具有不同耐药位点,1个对药物敏感,提取HIV-1病毒RNA,经逆转录PCR和巢式PCR获得3 100bp的pol区基因;将目的基因与pMDTM19-T载体连接后进行基因克隆,从阳性菌液中提取回收重组质粒并测序鉴定,最后评估考核品的均匀性和稳定性。结果 5个目的基因扩增测序均成功,与载体连接后得到重组载体V1、V2、V3、V4、V5,梯度退火荧光PCR结果显示考核品退火温度为64.1℃;质粒稀释倍数≤10 000时,各个样本做荧光PCR的Ct值均小于30,因此,将每种质粒稀释10 000倍分装制成考核品;选择浓度不同的3个样本V1、V2和V3分别做均匀性检测,3组Ct值差异均无统计学意义(P>0.05),该考核品均匀性良好;选择V1、V2、V3做稳定性检测,结果显示该考核品至少可以在室温和4℃稳定保存20d,在-20℃和-80℃保存反复冻存5次后浓度均未发生明显变化,表明该考核品稳定性较好。结论本研究中用克隆载体制备的HIV-1基因型耐药检测能力验证考核品均匀性稳定性良好,是一种经济便捷的HIV-1基因型耐药检测考核品制备方法 。
Objective To prepare HIV-1 genotypic drug resistance testing proficiency test samples and assess their homogeneity and stability. Methods Five HIV-1 positive plasma samples were detected in the previous study. Four of them had different drug resistance sites and one was drug-sensitive. HIV-1 viral RNA was extracted and 3 100 bp pol gene. The target gene was cloned into pMDTM19-T vector for gene cloning. The recombinant plasmid was extracted from the positive bacterium solution and sequenced. Finally, the homogeneity and stability of the samples were evaluated. Results The 5 target genes were successfully amplified and sequenced, and the recombinant vectors V1, V2, V3, V4 and V5 were obtained after being ligated with the vector. The gradient annealing fluorescence PCR results showed that the annealing temperature was 64.1 ℃. When the dilution of plasmid was less than 10 000, For each sample, the Ct values of fluorescent PCR were all less than 30, therefore, each plasmid was diluted 10,000 times and made into test articles. Three samples V1, V2 and V3 with different concentrations were selected for homogeneity test. Three groups of Ct (P> 0.05). The stability of the test items was good. The stability test of V1, V2 and V3 was done. The results showed that the test items could be stably stored for at least 20 days at room temperature and 4 ℃, ℃ and -80 ℃ after repeated cryopreservation five times did not change significantly after concentration, indicating that the assessment of good stability. Conclusions The HIV-1 genotype resistance test in this study is a good and stable method for HIV-1 genotypic drug resistance testing.