| AIDS seriously threatened the health of the human being. Although many achievements in HIV-1 diagnosis and treatment have been made in recent years, there was still an urgent need to pay more efforts on research on the diagnosis and treatment, so as to suppress the spread of AIDS in the world.HIV-1 diagnostic assays of HIV-1 relates to HIV-1 RNA loads, HIV-1 structural proteins and anti-HIV-1 antibody. These assays play an important role in the early diagnosis, and HIV-1 RNA loads detection techniques among them are most sensitive. HIV-1 RNA loads detection contributed to monitoring of viral replication during HIV-1 window period, the early diagnosis of vertical transmission, and HARRT in treatment of patients. However, HIV-1 RNA loads kits, have been approved by FDA, are much expensive. These kits contain Roche Amplicor HIV-1 Monitor Test, version 1.5, bioMerieux NucliSens HIV-1 QT Assay, and Versant HIV-1 RNA 3.0 Assay (bDNA)[4 Meanwhile, there is no domestic literature about HIV-1 p24 TaqMan FQ-PCR reported. Hence, the cost of these Kits is a burden for research on HIV-1 in some areas of limited resources. Therefore, it is essential to establish methods for detection of HIV-1 p24 loads, which should be commercial, high sensitive, specific, and stable.Progress in AIDS treatment can not bring in curative treatment. Additionally, the growing number of resistant strains and the serious drug side effects bring a more serious challenge to HIV-1 treatment. So we should establish an in vitro HIV-1 infectivity assay system for anti-HIV-1 drug screening, accelerating the process of research on AIDS.Part 1:Establishment and evaluation of a TaqMan FQ-PCR-based HIV-1 diagnostic assay systemObjective:TaqMan fluorescence quantitative polymerase chin reaction technology (HIV-1 p24 TaqMan FQ-PCR) was established for the detection of HIV-1 p24 loads. This diagnostic assay system can be applied in analyzing samples of clinical and laboratory.Methods:The pEGFP-N1-p24 was obtained through RNA extraction from H9 cell lines, reverse reaction, PCR and construction of plasmid, from which a fraction (a 154bp-lenghth fragment of p24) was amplified as standard products for establishing HIV-1 p24 TaqMan FQ-PCR. Samples of peripheral blood from 50 HIV-positive patients,30 healthy human,50 HBV-positive patients,50 HCV-positive patients and H9 cell lines (5.0×105/ml,0.5ml), pEGFP-Nl-p24 293T cells(5.0×105/ml,0.5ml) were detected by HIV-1 p24 TaqMan FQ-PCR and domestic TaqMan HIV-1 loads test kit and Genscreen plus HIV Ag-Ab kit (Bio-Rad). These assays were repeated in triplicate to verify the stability, sensitivity, specificity, reliability of HIV-1 p24 TaqMan FQ-PCR.Results:(1) All samples of peripheral blood from 50 HIV-positive patients, H9 cell line(5.0×105/ml,0.5ml) and pEGFP-Nl-p24 293T cells(5.0×10~5/ml,0.5ml) were HIV positive by Genscreen plus HIV Ag-Ab kit(Bio-Rad). All samples of 30 healthy human, 50 HBV-positive patients and 50 HCV-positive patients were HIV-negative by Genscreen plus HIV Ag-Ab kit (Bio-Rad)(2) HIV-1 positive rate was 100%, HIV-1 false positive rate was 0% by detection of peripheral blood of 50 HIV-1-positive patients through the p24 TaqMan FQ-PCR system or domestic TaqMan HIV-1 loads test kit. These results demonstrated the high sensitivity and specificity of HIV-1 p24 TaqMan FQ-PCR.(3) Difference between the average HIV-1 viral load detected by HIV-1 p24 TaqMan FQ-PCR and domestic TaqMan HIV-1 loads test kit was no statistically significant (t=-0.346, P= 0.730). The regression liner equation was:Log domestic TaqMan HIV-1 loads test kit copies/mL= 0.163+0.974×log HIV-1 p24 TaqMan FQ-PCR copies/mL.The correlation coefficient of quantification of two assay was 0.987(P<0.01). The results demonstrated the reliability of HIV-1 p24 TaqMan FQ-PCR was good.(4) Coefficient of variation (CV) of HIV-1 p24 loads between duplicated-wells of samples in signal experiment or means values of duplicated-wells in triplicated experiments were less than 5.09% through HIV-1 p24 TaqMan FQ-PCR. The results demonstrated the stability of HIV-1 p24 TaqMan FQ-PCR was good.Conclusion:HIV-1 p24 TaqMan FQ-PCR technique is reliable, high sensitive, specific, stable, economical, and can be initially used for detecting p24 loads of laboratory and clinical samples.Part 2:Establishment and evaluation of an in vitro HIV-1 infectivity assay systemObjective:An in vitro HIV-1 infectivity assay system was established. This system could be used in the preliminary application for anti-HIV-1 drug screening.Methods:JLTRG cells and H9 cells are mixed at different proportion for culture, of which the levels of EGFP expression in JLTRG are observed at 24,48,72,96 hours by fluorescence microscopy or by flow cytometry to assess infected JLTRG degree in the system. Optimal system which is used to screen anti-HIV-1 drugs.Results:(1) Most EGFP production were expressed JLTRG, most JLTRG were infected by HIV-1 which was released by H9, when JLTRG/H9 ratio was 10:1 in JLTRG/H9 mixed culture system. So infection rate was most high at the ratio in this system. (2) T-20 suppressed EGFP production of JLTRG in a dose-dependent manner in the system while JLTRG/H9 ratio was 10:1. The data shows that T-20 can effectively protect JLTRG from HIV-1 infection.Conclusions:An in vitro HIV-1 infectivity assay system could be used for anti-HIV-1 drugs screening. |
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