本文選題：獲得性免疫缺陷綜合征 + HIV-1��； 參考：《中國(guó)疾病預(yù)防控制中心》2014年碩士論文

【摘要】：第一部分三種HIV-1基因型耐藥檢測(cè)方法的一致性評(píng)價(jià) 目的 比較in-house HIV-1基因型耐藥檢測(cè)方法與TRUGENETM、ViroSeqTM兩種商品化基因耐藥檢測(cè)方法結(jié)果的一致性。 方法 1.取25份2009—-2013年來(lái)自美國(guó)Rush大學(xué)的HIV-1基因型耐藥檢測(cè)國(guó)際考核血漿樣本,分別使用in-house、TRUGENETM和ViroSeqTM三種HIV-1基因型耐藥檢測(cè)方法進(jìn)行檢測(cè),對(duì)其耐藥相關(guān)突變位點(diǎn)及對(duì)17種藥物的耐藥性進(jìn)行一致性評(píng)價(jià)。 2.取15份來(lái)自中國(guó)疾病預(yù)防控制中心性病艾滋病預(yù)防控制中心的待檢血漿樣本用in-house和TRUGENETM兩種方法補(bǔ)充證實(shí)所得結(jié)論,對(duì)兩種方法所得檢測(cè)結(jié)果進(jìn)行一致性評(píng)價(jià)。 結(jié)果 1.耐藥相關(guān)突變位點(diǎn)檢測(cè)結(jié)果的一致性評(píng)價(jià)：在25份國(guó)際考核樣品中,三種方法對(duì)耐藥相關(guān)突變位點(diǎn)檢測(cè)結(jié)果的一致率為99.42%(2933/2950),兩兩間比較一致性均為極強(qiáng)(Kappa值均0.81,P值均0.01)。 2.耐藥報(bào)告檢測(cè)結(jié)果的一致性評(píng)價(jià)：三種方法兩兩間比較的不一致結(jié)果主要集中在核苷類逆轉(zhuǎn)錄酶抑制劑的齊多夫定(zidovudine, AZT)、去羥肌苷(didanosine, ddl)、司他夫定(stavudine, d4T)、阿巴卡韋(abacavir, ABC)4種藥物,且主要為輕微不一致。其中in-house與ViroSeq方法兩兩比較結(jié)果的輕微不一致率分別為28%(7/25)、28%(7/25)、16%(4/25)、20%(5/25)；in-house與TRUGENETM的輕微不一致率分別為44%(11/25)、28%(7/25)、36%(9/25)、28%(7/25);TRUGENETM與ViroSeqTM的輕微不一致率分別為24%(6/25)、8%(2/25)、28%(7/25)、8%(2/25)。In-house與ViroSeqTM比較時(shí)藥物AZT,In-house與TRUGENETM比較時(shí)藥物ddI、d4T、ABC、替諾福韋(Tenofovir,TDF)兩兩比較的結(jié)果為中度一致(加權(quán)Kappa系數(shù)分別為0.54,0.44,0.52,0.42,0.59,P值均0.01),其余的兩兩比較結(jié)果均為高度一致(加權(quán)Kappa系數(shù)為0.61-0.80)或一致性極強(qiáng)(加權(quán)Kappa系數(shù)0.80)。 3.15種待檢血漿樣本耐藥相關(guān)突變位點(diǎn)的一致率為99.55%(1762/1770),17種藥物耐藥結(jié)果兩兩比較的不一致也主要集中在AZT、ddI、d4T、ABC四種藥物。 結(jié)論 In-house HIV-1基因型耐藥檢測(cè)方法與TRUGENETM、ViroSeqTM兩種商品化的基因型耐藥檢測(cè)方法在耐藥檢測(cè)上具有較好的一致性。 第二部分HIV-1基因型耐藥檢測(cè)能力驗(yàn)證考核品的制備和評(píng)估 目的 制備in-house HIV-1基因型耐藥檢測(cè)能力驗(yàn)證考核品,并對(duì)其均勻性和穩(wěn)定性進(jìn)行評(píng)估。 方法 1.從陽(yáng)性血漿中提取HIV-1病毒核酸,經(jīng)逆轉(zhuǎn)錄PCR和巢式PCR獲得3100bp的pol區(qū)基因。 2.將目的基因與pMDTM19-T載體連接,將重組載體導(dǎo)入JM109感受態(tài)細(xì)胞,將其接種于涂有Amp(氨芐霉素)和X-Gal的LB瓊脂培養(yǎng)基上培養(yǎng)。從培養(yǎng)基中挑取白色菌落接種于LB肉湯振蕩過(guò)夜培養(yǎng),PCR鑒定,從陽(yáng)性菌液中提取回收重組質(zhì)粒并測(cè)序鑒定。 3.查閱文獻(xiàn)資料確定熒光PCR的引物、反應(yīng)體系,并用梯度退火方法確定退火溫度。 4.用陰性血漿稀釋重組質(zhì)粒,根據(jù)熒光PCR方法檢測(cè)的Ct值確定最適濃度。稀釋分裝,制備考核品。 5.從重組質(zhì)粒稀釋樣本選擇濃度不同的三個(gè)樣本,評(píng)估樣本的均勻性。 6.將三個(gè)不同濃度的樣本,評(píng)估樣本的穩(wěn)定性。 結(jié)果 1.擴(kuò)增測(cè)序得到5個(gè)目的基因,分別編號(hào)為01、02、03、04、05。 2.目的基因與載體連接,得到重組載體V1、V2、V3、V4、V5。 3.梯度退火熒光PCR結(jié)果顯示,熒光PCR的退火溫度為64.1℃。 4.樣本稀釋實(shí)驗(yàn)結(jié)果表明,質(zhì)粒稀釋倍數(shù)≤10000時(shí),各個(gè)樣本做熒光PCR的Ct值均小于30,因此,將每種質(zhì)粒稀釋10000倍分裝制成考核品。 5.選擇濃度不同的三個(gè)樣本V1、V2、V3,做均勻性檢測(cè)。兩組Ct值方差齊性檢測(cè)結(jié)果不具有顯著性差異(P均0.05),認(rèn)為重組質(zhì)粒稀釋制成的考核品均勻性良好。 6.選擇V1、V2、V3做穩(wěn)定性檢測(cè),結(jié)果顯示：重組質(zhì)粒構(gòu)建的考核品至少可以在室溫和4℃C穩(wěn)定保存20天；在-20℃C和-80℃C保存反復(fù)凍存5次濃度均未發(fā)生明顯變化。 結(jié)論 本研究中用克隆載體制備的in-house HIV-1基因型耐藥檢測(cè)能力驗(yàn)證考核品均勻性穩(wěn)定性良好,該方法解決了耐藥樣本難以大量獲得的難題,是一種經(jīng)濟(jì)便捷的in-house HIV-1基因型耐藥檢測(cè)考核品制備方法。
[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.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R392

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本文編號(hào)：2066388