Establishment and optimization of a high throughput phenotypic test for the detection of drug resist-ance in human immunodeficiency virus(HIV)strains
10.3760/cma.j.issn.0254-5101.2014.12.009
- VernacularTitle:基于假病毒的高通量人类免疫缺陷病毒表型耐药性检测方法的建立和优化
- Author:
Jianhui NIE
;
Sihong XU
;
Aijing SONG
;
Juan ZHAO
;
Qingqing CHEN
;
Jian MA
;
Weijin HUANG
;
Youchun WANG
- Publication Type:Journal Article
- Keywords:
Human immunodeficiency virus;
Drug resistance;
Phenotypic test
- From:Chinese Journal of Microbiology and Immunology
2014;(12):941-949
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a high throughput phenotypic test for the detection of drug re-sistance in human immunodeficiency virus(HIV)strains. Methods The gene encoding luciferase was in-activated 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 in-to a new backbone plasmid through infusion. The factors that might affect the results of the test were opti-mized. 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 tran-scriptase. 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 pseud-ovirus test. The results of the two tests presented good consistency. Conclusion The high throughput phe-notypic 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 antiretrovi-ral therapy in a rapid and effective way.
