Design of polymerase chain reaction primers for the selective amplification of HIV-1 RNA in the presence of HIV-1 DNA.
Design of polymerase chain reaction primers for the selective amplification of HIV-1 RNA in the presence of HIV-1 DNA.
复制标题
设计聚合酶链式反应引物，用于在 HIV-1 DNA 存在的情况下选择性扩增 HIV-1 RNA。
DOI：
10.1097/00002030-199206000-00004
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Goldstein,H
中科院分区：
文献类型：
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作者：
Kollmann,TR;Zhuang,X;Rubinstein,A;Goldstein,H
设计：逆转录后，通过聚合酶链式反应(PCR)扩增检测HIV-1RNA的存在。由于HIV-1RNA的特异性检测可能会因微量的HIV-1DNA污染而变得复杂，因此样品在分析之前要用脱氧核糖核酸酶(DNase)处理。这一额外的步骤增加了RNA降解和样品污染的可能性。方法：设计了一种名为HG141的引物，用于与存在于HIV-1基因组中的聚(A)链和所有HIV-1信使(M)RNA及其5‘端与聚(A)链上游3’端的区域杂交。HG141引物/HIV-1RNA或互补(C)DNA复合体的稳定性增加，使得HG141和返回的引物HG62可以在高于引物-HIV-1DNA复合体熔化温度(Tm)的温度下进行PCR扩增。结果：从感染HIV-1的H9细胞中获得的样品反转录后，HG62/141-Primed PCR扩增特异性地检测到HIV-1 RNA序列，而不需要DNA酶预处理。结论：由于HIV-1RNA的存在预示着HIV-1vrion的存在，所述的RNA特异性引物有助于评估HIV-1感染者的HIV-1复制和血浆HIV-1病毒载量。这在评估治疗干预措施对艾滋病毒-1感染的效果方面应该是有用的。
Design: Following reverse transcription, the presence of HiV-1 RNA can be detected by polymerase chain reaction (PCR) amplification. Since specific detection of HIV-1 RNA may be complicated by contamination with minute quantities of HIV-1 DNA, samples are treated with deoxyribonuclease (DNase) prior to analysis. This additional step increases the possibility of RNA degradation and sample contamination.Methods: A primer, HG141, was designed to hybridize to the poly (A) tract present in HIV-1 genomic and all HIV-1 messenger (m) RNA with its 5'end and to the region upstream of the poly (A) tract with its 3'end. The increased stability of the HG141 primer/HIV-1 RNA or complementary (c) DNA complex, enabled PCR amplification to be performed with HG141 and the return primer HG62 at an annealing temperature above the melting temperature (Tm) of the primer-HIV-1 DNA complex.Results: After reverse transcription of samples obtained from HIV-1-infected H9 cells, HG62/141-primed PCR amplification specifically detected HIV-1 RNA sequences without the need for DNase pre-treatment. This technique was more sensitive for the detection of HIV-1 RNA than SK38/39-primed PCR amplification of DNase-treated samples.Conclusions: Since the presence of HIV-1 RNA is indicative of HIV-1 replication for the presence of HIV-1 vjrions, the RNA-specific primer described should facilitate the assessment of HIV-1 replication and the plasma HIV-1 viral load in HIV-1-infected individuals. This should prove useful in the evaluation of the effects of therapeutic interventions on HIV-1 infection.