To construct a stable HIV-1 nucleic acid quantitative detection of internal standard based on virus particles.

p24 region was mutated without changing p24 conservative district and inserted into phenotype resistant vector pcDNA6.2-LTR gagpol by PCR amplification method, following by the construction of pNL43-?ENV-LUC expression vector containing the specific mutation base by Gateway recombinant technology. This vector and VSV plasmid were cotransfected into 293T cell lines and HIV-1 pseudotyped virus was collected from the supernatant 48 hours later. The loads of HIV-1 were quantified by detection of p24 antigen titers. The multiproportion diluted liquids of the internal standard plasmid, the mixture of the internal standard plasmid and positive samples and the mixture of the internal standard pseudotyped viral particles and positive samples were used as the template for the real-time quantitative PCR to verify whether the nucleic acids levels matched the probes of internal standard plasmid, and whether they could be interfered by the sample probe and primers.

The fragment of specific site mutation in p24 conservative region was inserted into the phenotype resistant vector pcDNA6.2-LTR gagpol. The detective p24 antigen was 283.2 pg/ml, while HIV-1 pseudotyped virus was 3.61 × 10⁹IU/ml. Internal standard plasmid and internal standard pseudotyped viral particles matched the probes of internal standard plasmid, which were confirmed using real-time quantitative PCR and not interfered by the sample probe and primers.

The novel internal standard construced with pseudotyped virus could be used to detect plasma HIV-1 level.