[Primers of gag gene for HIV-1 subtyping in China and application thereof in practice].
OBJECTIVE
To design the appropriate primers of gag gene for HIV-1 subtyping in molecular epidemiology survey based on the genetic characteristics derived from the main HIV-1 strains prevailing in China.


METHODS
The gag genes of HIV-1 CRF07_BC, CRF08_BC, and subtype C, together with subtyping reference sequences, were obtained from HIV sequence database. Referring to the alignments and genetic characteristics of HIV-1 full gag sequences, new primers of gag gene for amplification and subtyping were designed. The target fragment was used to construct neighbor-joining phylogenetic tree and evaluate its reliability. The newly designed primers (GUX/GDX) were used to amplify the plasma samples to evaluate their efficiency.


RESULTS
The phylogenetic tree of 306/c-gag fragments (positions 836-1507 of HIV-1 strain HXB2) showed that CRF07_BC and subtype C strains formed clusters with low bootstrap values (59% for CRF07_BC and 70% for subtype C), and the phylogenetic tree could not distinguish the sequences of CRF07_BC, CRF08_BC, and subtype C very well. Whereas the sequences of CRF07_BC, CRF08_BC, and subtype C from GUX/GDX (positions 781-1861) were clustered separately with higher bootstrap values (99%, 99%, and 77% respectively). In practice, a very good amplification and sequencing efficiency with over 90% positive results on average were obtained with GUX/GDX. Five clusters of subtype B, C, CRF01_AE, CRF07_BC, and CRF08_BC were formed with higher confidence (Bootstrap values all above 80%). The reliable phylogenetic tree could be constructed based on the fragments sequenced only with antisense primer (GDX).


CONCLUSIONS
Fragments obtained with GUX/GDX primers of gag gene can be used to reconstruct phylogenetic tree with high reliability to distinguish the HIV-1 strains circulating in China, especially for the major BC recombinant and subtype C strains, which provides a useful tool in HIV molecular epidemiologic research.
Xin
Ruo-lei
RL
State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention, China Center for Diseases Prevention and Control, Beijing 100050, China.
Feng
Yi
Y
Cheng
Chun-lin
CL
Hu
Yuan-yuan
YY
Meng
Zhe-feng
ZF
Xing
Hui
H
Shao
Yi-ming
YM
He
Xiang
X
D002681:China / Q000453:epidemiology; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D015750:Genes, gag / Q000235:genetics*; D016679:Genome, Viral; D005838:Genotype; D005843:Geography; D015658:HIV Infections / Q000453:epidemiology / Q000821:virology*; D015497:HIV-1 / Q000145:classification* / Q000235:genetics*; D006801:Humans; D010802:Phylogeny; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004279:DNA, Viral