| ObjectivesTo develop a simple and practical real-time PCR assay for HIV-1 variants prevailing in Guangxi.Methods1. Design of the primers and Taqman probe HIV-1 sequences prevailing in Guangxi was downloaded from the Los Alamos National Laboratory website(http://hiv-web.lanl.gov), and consensus sequences were obtained by alignment of these sequences using Clustalx and Bioedit software. Then the primers and Taqman probe were designed with primer Express software (Applied Biosystems).2.Construction of the standard The target sequence was amplified from a HIV-1 positive person from Guangxi by RT-PCR, then it was cloned into the plasmid (pMD18-T) cloning vector and transfected into E. coli (JM 109). The correct recombinant plasmid was selected using Blue-White Selection, enzyme digestion and sequencing and was used as standard after quantification and serial dilution.3. Methodological evaluation The real-time assay was developed by using the designed Oligonucleotides, the PCR system and the reaction temperature were optimized. Thereafter, the liner ranger, the low detection limit, specificity and reproducibility of the method were evaluated.4.Comparison with the NASBA method 56 HIV-1 positive samples were detected by the real-time PCR and NASBA method respectively in order to assess the consistency and correlation of the two kinds of methods.5. Evaluation the suitability of subtype diversity The suitability of subtype diversity was assessed by comparing the detection results of viral loads among different subtypes of the 56 HIV-1 samples.6. Evaluation using international references 15 international references of unknown subtype and concentration were detected by the real-time PCR with double blind to assess the accuracy and suitability.7. Comparison with the domestic method The domestic HIV-1 real-time PCR Kit(PG Biotech)was employed to quantitate the viral load of 72 samples. Meanwhile, 72 samples were also detected by our real-time PCR assay to test the consistency of two kinds of real-time PCR assay.Results1. Design of the primers and Taqman probe The designed primers and probe was suitable for amplify the HIV-1 variants prevailing in Guangxi.2. Construction of the standard The correct recombinant plasmid containing the target sequence was constructed, the standard were 10-fold serial diluted and were used to produce the standard curve.3. Methodological evaluation The low detection limit of real-time PCR was 150 copies per milliliter. When the range of external standard was 1.5×10~2-1.5×10~8 copies per milliliter, the correlation coefficient of standard between the input copies and fluorescence intensity was 0.9968, the slope of standard curve was -3.384. The specificity of the real-time PCR was 100%. The intra-assay reproducibility CV were 0.977%, 1.129%, 1.415%, 1.328%, 0.528%, 0.798% respectively, the inter-assay reproducibility CV was 1.156%. The reactive reagents repeated freeze thaw cycles also demonstrated a good perfomance as well as the fresh one.4. Comparison with the NASBA method The average HIV viral load detected by real-time PCR and NASBA was (4.411±0.673) log copies/ ml and (4.467±0.658)log copies/ml respectively, and the difference was no statistically significant between the two groups (t=1.207,P=0.233>0.05). Both the real-time PCR assay and NASBA method have the satisfactory positive rates and the similar viral load rank(P>0.05). The correlation coefficient of quantification between real-time PCR and NASBA was 0.880(P<0.01), the regression liner equation is: logNASBAcopies/mL =0.671+0.861×log real-time PCR copies/mL.5. Evaluation the suitability of subtype diversity The real-time PCR assay was suitable for subtype B', CRF01-AE, CRF07-BC and CRF08-BC respectively, and the positive rates were similar for different subtypes.6. Evaluation using international references The detection result of 15 references indicated the real-time PCR method could quantitate the samples with virus load greater than 103 copies per milliliter, however, the samples below the 103 copies number can not accurately quantitated.7. Comparison with the domestic method The average HIV viral load detected by our real-time PCR and real-time PCR Kit(PG Biotech) was (4.393±0.690) log copies/ml and (4.330±0.809)log copies/ml respectively, and the difference was no statistically significant between the two groups (t=-1.438, P=0.156>0.05). The domestic HIV-1 real-time PCR Kit quantitated 63 out of the 72 samples (87.5%), and our real-time PCR assay can detect 61 samples (84.7), there were no statistical difference in positive rate and viral load rank between two kinds of real-time PCR assay (P>0.05). The regression liner equation was: Log PG real-time PCR copies/mL= -0.388+1.074×log our real-time PCR copies/mL.The correlation coefficient of quantification of two assay was 0.917 (P<0.001). In addition, when the samples with viral load between 103 and 105 copies per milliliter, two kinds of real-time PCR kept a perfect consistency (37/43), whereas two methods of quantitative analysis given goodish perfomance while the viral load beyond 105 or below 103 copies.ConclusionWe have developed a new real-time PCR assay. The method can detect a wide liner range of HIV-positive samples with a high specificity, a reasonbale reproducibility, a considerable sensitivity, and a good reagent stability. Furthermore, the real-time PCR method has perfect correlation with NASBA, and keep satisfactory consistency with the domestic HIV-1 real-time PCR Kit. It is suitbable for different subtypes prevailing in Guangxi, consequently, this method is scientific, practical and will be of great potential applications in viral load monitoring, early infection detection, and in monitoring of drug resistance during ART treatment.
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