RNA与前病毒DNA进化分析技术在判定HIV-1传播关系中的作用比较

Comparison of RNA and proviral DNA phylogenetic analysis in the evaluation of HIV-1transmission

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 compare the amplification efficiency using templates of HIV-1 RNA and proviral DNA and the evaluation of the transmission between couples by genetic linkage analysis. Methods HIV-1RNA and proviral DNA were extracted from the HIV-1sero-positive couples collected in the National HIV-1Sample Bank.The HIV-1pol gene fragments were amplified by nested polymerase chain reaction(nPCR),and the genetic linkage was evaluated using phylogenetic and genetic distance analysis.Meanwhile,the transmission direction was evaluated from the follow-up information. Results A total of 42(42/211,19.91%)pol sequences obtained using RNA as the template from 211 HIV-1plasma couples and sequences from 12 couples were obtained.Final linkage analysis found 10 out of 12couples(10/12,83.33%)were linked,1of 12(1/12,8.33%)were unlink,and 1 of 12(1/12,8.33%)was undetermined.There were 232(232/270,85.93%)pol sequences obtained when using proviral DNA as templates from 270 HIV-1 whole blood samples.The 232 sequences include those from 97 couples,final linkage status were determined for 86 out of 97couples(86/97,86.66%),7of 97(7/97,7.22%)were unlinked,while 4of 97(4/97,4.12%)were undertermined.The Bootstrap values of the two phylogenetic trees were significantly correlated(r = 0.86,P=0.001),while the difference of genetic distance was not statistically significant(Z=-8.10,P=0.443).The genetic distance of the linkage couples were 0.009 2and 0.011 0between the amplified sequences using RNA and proviral DNA as the amplification templates. Conclusions Using proviral DNA as template could improve the efficiency of amplification,especially when the couples already started antiviral therapy.To some extent,proviral DNA can replace RNA in amplification to evaluate the transmission relationships in seropositibe couples.The average genetic distance of linked couples amplified by proviral DNA is 0.011 0,and it could serve as a reference for evaluating the transmission relationships in HIV-1sero-positive couples.