Abstract: (17 Views)
A B S T R A C T
Background and Objectives
HTLV-1 is the first identified human oncogenic retrovirus, associated with ATL and HAM/TSP diseases. The HBZ gene, one of the key genes of this virus, plays an important role in disease progression due to its low immunogenicity and genetic stability. Despite the progress of diagnostic methods such as ELISA and Western blot, indeterminate results are occasionally reported. Moreover, measuring viral load is critical for prognosis of the disease. In this study, a sensitive system for determining HTLV-1 proviral load based on the HBZ gene was designed and implemented using the TaqMan Real Time PCR method.
Materials and Methods
In this experimental study, primers and probes were designed to amplify a 106 bp region of the HBZ gene. Then, genomic DNA was extracted from PBMCs. After amplification of the HBZ gene, the PCR product was inserted into the TOPO TA vector and used as a standard to develop the TaqMan Real Time PCR method.
Results
The logarithmic dilutions of 1.2×101-108 were prepared from the recombinant plasmid to draw the standard curve. The slope of the standard curve line was -3.2 and R2 was 0.97, indicating the linearity and efficiency of the test. The results of the reproducibility of the reaction at the intra-assay and inter-assay levels (three replicates per step) and the average coefficient of variation CV of the standard samples were below 5.3% and 5.9%, respectively. Also, the Limit of Detection of this test was determined to be 1.2×101 Copies/µL, reflecting the high sensitivity of this assay.
Conclusions
Given its high sensitivity, rapid analysis and ease of use, the Real Time PCR method represents a suitable approach for determining the proviral load of HTLV-1 and assessing the prognosis of associated diseases.
Background and Objectives
HTLV-1 is the first identified human oncogenic retrovirus, associated with ATL and HAM/TSP diseases. The HBZ gene, one of the key genes of this virus, plays an important role in disease progression due to its low immunogenicity and genetic stability. Despite the progress of diagnostic methods such as ELISA and Western blot, indeterminate results are occasionally reported. Moreover, measuring viral load is critical for prognosis of the disease. In this study, a sensitive system for determining HTLV-1 proviral load based on the HBZ gene was designed and implemented using the TaqMan Real Time PCR method.
Materials and Methods
In this experimental study, primers and probes were designed to amplify a 106 bp region of the HBZ gene. Then, genomic DNA was extracted from PBMCs. After amplification of the HBZ gene, the PCR product was inserted into the TOPO TA vector and used as a standard to develop the TaqMan Real Time PCR method.
Results
The logarithmic dilutions of 1.2×101-108 were prepared from the recombinant plasmid to draw the standard curve. The slope of the standard curve line was -3.2 and R2 was 0.97, indicating the linearity and efficiency of the test. The results of the reproducibility of the reaction at the intra-assay and inter-assay levels (three replicates per step) and the average coefficient of variation CV of the standard samples were below 5.3% and 5.9%, respectively. Also, the Limit of Detection of this test was determined to be 1.2×101 Copies/µL, reflecting the high sensitivity of this assay.
Conclusions
Given its high sensitivity, rapid analysis and ease of use, the Real Time PCR method represents a suitable approach for determining the proviral load of HTLV-1 and assessing the prognosis of associated diseases.
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