Establishment and optimization of a high throughput phenotypic test for the detection of drug resist-ance in human immunodeficiency virus（HIV）strains
Objective To establish a high throughput phenotypic test for the detection of drug resistance in human immunodeficiency virus（HIV）strains. Methods The gene encoding luciferase was inactivated through restriction enzyme digestion and ligation. LacZ gene was used to replace the genes encoding original protease and reverse transcriptase. pol genes were amplified from pSG3△env plasmid and cloned into a new backbone plasmid through infusion. The factors that might affect the results of the test were optimized. Results The parental backbone plasmid pNL4-3. Lac was constructed,of which the gene encoding luciferase was inactivated and bearing the LacZ gene instead of genes encoding protease and reverse transcriptase. Several influential factors including cell numbers（10 000 / well）,virus inoculation（200 TCID50 /well）and the concentration of DEAE-dextran（15 μg/ ml）were optimized. The reproducibility of this test was confirmed by testing 12 anti-HIV drugs against 2 pseudovirus strains 8 times,presenting the coefficient of variations（CVs）from 4. 32% to 28. 46% . Six types of pseudovirus were constructed and tested against the 12 anti-HIV drugs,the results of which were compared with those by using the pSG3△env-based pseudovirus test. The results of the two tests presented good consistency. Conclusion The high throughput phenotypic test based on pNL4-3. Lac plasmid,combining the advantages of pSG3△env and pNL4-3 systems, could be used to analyze the drug resistance patterns of HIV-1 infectors and screen new drugs for antiretroviral therapy in a rapid and effective way. 
 
Key words: 
Human immunodeficiency virus; Drug resistance; Phenotypic test