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目的分别以HIV-1RNA和前病毒DNA为模板进行扩增实验,比较不同实验方法对判断HIV-1阳转家庭基因关联性的效率。方法对国家分析库中确认的阳转家庭,分别提取HIV-1RNA和前病毒DNA,用巢式PCR扩增pol区基因,通过系统进化树和基因距离分析配对家庭基因关联性。结果以RNA为模板扩增211份HIV-1阳性血浆,获得42份(42/211,19.91%)基因序列。其中共有12对配对家庭,确定有传播关系10对(10/12,83.33%),确定无传播关系1对(1/12,8.33%),不确定是否有传播关系1对(1/12,8.33%);以前病毒DNA为模板扩增270份HIV-1阳性全血,获得232份(232/270,85.93%)基因序列。其中共有97对配对家庭,确定有传播关系86对(86/97,88.66%),确定无传播关系7对(7/97,7.22%),不确定是否有传播关系4对(4/97,4.12%)。以RNA和前病毒DNA为模板扩增的序列系统进化树Bootstrap值高度相关(r=0.86,P=0.001),基因距离差异无统计学意义(Z=-8.10,P=0.443)。结论以前病毒DNA为模板能显著提高扩增阳性率,特别是在抗病毒治疗家庭中。前病毒DNA在一定程度上可以代替RNA为模板进行扩增,判断家庭内传播的关系。
OBJECTIVE: To amplify HIV-1 RNA and provirus DNA respectively, and to compare the efficiency of different experimental methods in determining the association of HIV-1 positive transgenic family genes. Methods HIV-1 RNA and provirus DNA were extracted from the positive transgenic families identified in the national analysis database. The nested PCR was used to amplify the pol region genes. The phylogenetic tree and genetic distance were used to analyze the association of matched genes. Results 211 HIV-1 positive plasma was amplified using RNA as a template, and 42 (42 / 211,19.91%) gene sequences were obtained. Among them, there are 12 pairs of matched families, 10 pairs (10/12, 83.33%) were confirmed to have communication relationship, 1 pair (1/12, 8.33%) were confirmed to have no communication relationship, 8.33%). A total of 232 HIV-1 positive whole blood samples were amplified by using the former viral DNA as a template. 232 (232 / 270,85.93%) gene sequences were obtained. Among them, there were 97 pairs of matched families, 86 pairs (86/97, 88.66%) were confirmed to have communication relationship, 7 pairs (7/97, 7.22%) were confirmed to have no communication relationship, 4 (4/97, 4.12%). The Bootstrap value of sequence phylogenetic tree based on RNA and proviral DNA was highly correlated (r = 0.86, P = 0.001). There was no significant difference in gene distance (Z = -8.10, P = 0.443). Conclusion Previous viral DNA as a template can significantly increase the positive rate of amplification, especially in anti-virus treatment of the family. Pro-virus DNA to some extent instead of RNA as a template for amplification, to determine the spread of family relationships.