HIV-1基因型耐药检测方法的评价及其能力验证考核品的制备和评估

发布时间：2018-06-25 14:55

本文选题：获得性免疫缺陷综合征 + HIV-1　；参考：《中国疾病预防控制中心》2014年硕士论文

【摘要】：第一部分三种HIV-1基因型耐药检测方法的一致性评价目的比较in-house HIV-1基因型耐药检测方法与TRUGENETM、ViroSeqTM两种商品化基因耐药检测方法结果的一致性。方法 1.取25份2009—-2013年来自美国Rush大学的HIV-1基因型耐药检测国际考核血浆样本,分别使用in-house、TRUGENETM和ViroSeqTM三种HIV-1基因型耐药检测方法进行检测,对其耐药相关突变位点及对17种药物的耐药性进行一致性评价。 2.取15份来自中国疾病预防控制中心性病艾滋病预防控制中心的待检血浆样本用in-house和TRUGENETM两种方法补充证实所得结论,对两种方法所得检测结果进行一致性评价。结果 1.耐药相关突变位点检测结果的一致性评价：在25份国际考核样品中,三种方法对耐药相关突变位点检测结果的一致率为99.42%(2933/2950),两两间比较一致性均为极强(Kappa值均0.81,P值均0.01)。 2.耐药报告检测结果的一致性评价：三种方法两两间比较的不一致结果主要集中在核苷类逆转录酶抑制剂的齐多夫定(zidovudine, AZT)、去羟肌苷(didanosine, ddl)、司他夫定(stavudine, d4T)、阿巴卡韦(abacavir, ABC)4种药物,且主要为轻微不一致。其中in-house与ViroSeq方法两两比较结果的轻微不一致率分别为28%(7/25)、28%(7/25)、16%(4/25)、20%(5/25)；in-house与TRUGENETM的轻微不一致率分别为44%(11/25)、28%(7/25)、36%(9/25)、28%(7/25);TRUGENETM与ViroSeqTM的轻微不一致率分别为24%(6/25)、8%(2/25)、28%(7/25)、8%(2/25)。In-house与ViroSeqTM比较时药物AZT,In-house与TRUGENETM比较时药物ddI、d4T、ABC、替诺福韦(Tenofovir,TDF)两两比较的结果为中度一致(加权Kappa系数分别为0.54,0.44,0.52,0.42,0.59,P值均0.01),其余的两两比较结果均为高度一致(加权Kappa系数为0.61-0.80)或一致性极强(加权Kappa系数0.80)。 3.15种待检血浆样本耐药相关突变位点的一致率为99.55%(1762/1770),17种药物耐药结果两两比较的不一致也主要集中在AZT、ddI、d4T、ABC四种药物。结论 In-house HIV-1基因型耐药检测方法与TRUGENETM、ViroSeqTM两种商品化的基因型耐药检测方法在耐药检测上具有较好的一致性。第二部分HIV-1基因型耐药检测能力验证考核品的制备和评估目的制备in-house 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 1.从阳性血浆中提取HIV-1病毒核酸,经逆转录PCR和巢式PCR获得3100bp的pol区基因。 2.将目的基因与pMDTM19-T载体连接,将重组载体导入JM109感受态细胞,将其接种于涂有Amp(氨苄霉素)和X-Gal的LB琼脂培养基上培养。从培养基中挑取白色菌落接种于LB肉汤振荡过夜培养,PCR鉴定,从阳性菌液中提取回收重组质粒并测序鉴定。 3.查阅文献资料确定荧光PCR的引物、反应体系,并用梯度退火方法确定退火温度。 4.用阴性血浆稀释重组质粒,根据荧光PCR方法检测的Ct值确定最适浓度。稀释分装,制备考核品。 5.从重组质粒稀释样本选择浓度不同的三个样本,评估样本的均匀性。 6.将三个不同浓度的样本,评估样本的稳定性。结果 1.扩增测序得到5个目的基因,分别编号为01、02、03、04、05。 2.目的基因与载体连接,得到重组载体V1、V2、V3、V4、V5。 3.梯度退火荧光PCR结果显示,荧光PCR的退火温度为64.1℃。 4.样本稀释实验结果表明,质粒稀释倍数≤10000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10000倍分装制成考核品。 5.选择浓度不同的三个样本V1、V2、V3,做均匀性检测。两组Ct值方差齐性检测结果不具有显著性差异(P均0.05),认为重组质粒稀释制成的考核品均匀性良好。 6.选择V1、V2、V3做稳定性检测,结果显示：重组质粒构建的考核品至少可以在室温和4℃C稳定保存20天；在-20℃C和-80℃C保存反复冻存5次浓度均未发生明显变化。结论本研究中用克隆载体制备的in-house HIV-1基因型耐药检测能力验证考核品均匀性稳定性良好,该方法解决了耐药样本难以大量获得的难题,是一种经济便捷的in-house HIV-1基因型耐药检测考核品制备方法。
[Abstract]:Part one: consistency evaluation of three methods for detection of HIV-1 genotype resistance
objective
To compare the results of in-house HIV-1 genotype resistance test with the results of two commercial gene resistance detection methods of TRUGENETM and ViroSeqTM.
Method
1. 25 HIV-1 genotypic resistance detection plasma samples were collected from Rush University in the United States from 2009 to -2013, and three HIV-1 genotypic resistance detection methods of in-house, TRUGENETM and ViroSeqTM were used to detect the resistance related mutation sites and the consistency of the resistance to 17 drugs.
2. samples of 15 samples from the center for STD and AIDS prevention and control center of China Disease Control and prevention center were supplemented by two methods of in-house and TRUGENETM, and the results of the two methods were evaluated in consistency.
Result
The consistency evaluation of the detection results of 1. resistance related mutation sites: of the 25 international assessment samples, the consensus rate of the three methods for the detection of resistance related mutation sites was 99.42% (2933/2950), and the consistency of the 22 comparison was very strong (Kappa value was 0.81, P value was 0.01).
The consistency evaluation of the results of 2. drug resistance reports: the inconsistencies between the three methods were mainly concentrated on zidovudine (zidovudine, AZT), didanosine (DDL), stavudine (d4T), and abacavir (ABC) of nucleoside reverse transcriptase inhibitors, and were mainly minor inconsistencies. The slight inconsistencies between in-house and ViroSeq 22 were 28% (7/25), 28% (7/25), 16% (4/25), 20% (5/25), and the slight inconsistencies between in-house and TRUGENETM were 44% (11/25), 28% (7/25), 36% (9/25), 28% (7/25), 24%, 8%, 28%, 8%, respectively. When compared with ViroSeqTM, the drug AZT, In-house and TRUGENETM were compared with ddI, d4T, ABC, and the results of the comparison of Nuo Fuwei (Tenofovir, TDF) 22 were moderately consistent (the weighted Kappa coefficient was 0.54,0.44,0.52,0.42,0.59, 0.01), and the other 22 comparison results were all highly consistent. Very strong (weighted Kappa coefficient 0.80).
The consistent rate of resistance related mutation sites in the 3.15 plasma samples was 99.55% (1762/1770), and the differences in the 17 drug resistance results 22 were also mainly concentrated in the AZT, ddI, d4T, and ABC four drugs.
conclusion
In-house HIV-1 genotypic resistance detection method and TRUGENETM, ViroSeqTM two commercialized genotypic resistance detection methods have good consistency in drug resistance detection.
The second part is the preparation and evaluation of HIV-1 genotype resistance testing proficiency test products.
objective
In-house HIV-1 genotypes were tested for their ability to detect drug resistance, and their uniformity and stability were evaluated.
Method
1. HIV-1 virus nucleic acid was extracted from positive plasma, and the pol gene of 3100bp was obtained by reverse transcription PCR and nested PCR.
2. the target gene was connected with the pMDTM19-T vector, and the recombinant vector was introduced into the JM109 receptive cell and inoculated on the LB agar medium coated with Amp (ampicillin) and X-Gal. The white colonies were inoculated from the medium in the culture medium for the overnight culture of LB broth, PCR identification, and the recovery of the recombinant plasmid from the positive bacteria and sequencing and identification.
3. the primers and reaction systems of fluorescent PCR were determined through literature review, and the annealing temperature was determined by gradient annealing.
4. the recombinant plasmid was diluted with negative plasma, and the optimum concentration was determined according to the Ct value detected by fluorescence PCR.
5. select three samples with different concentrations from the recombinant plasmid dilution samples to evaluate the uniformity of the samples.
6. three different concentrations of samples were used to evaluate the stability of the samples.
Result
1. amplified and sequenced, 5 target genes were obtained, numbering 01,02,03,04,05. respectively.
2. the target gene was connected with the vector to obtain recombinant vector V1, V2, V3, V4, V5.
3. the results of gradient annealing PCR show that the annealing temperature of the fluorescent PCR is 64.1 C.
The 4. sample dilution test showed that the Ct value of each sample was less than 30 when the plasmid dilution multiple was less than 10000, so that each plasmid was diluted by 10000 times to prepare the PCR.
5. the three samples with different concentration V1, V2 and V3 were selected for uniformity detection. There was no significant difference between the two groups of Ct value variance homogeneity test (P 0.05).
6. V1, V2 and V3 were selected for stability detection. The results showed that the assessment products constructed by recombinant plasmid could be preserved at at least 20 days at room temperature and 4 centigrade C, and there was no significant change in 5 concentrations at -20 C and -80 C.
conclusion
In this study, the resistance detection ability of in-house HIV-1 genotypes prepared by cloned carrier is good for the stability of uniformity. This method solves the difficult problem that the drug resistance samples are difficult to obtain. It is an economical and convenient method for the preparation of in-house HIV-1 genotypic resistance detection assessment products.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392

【参考文献】