Cells and gDNA preparation
Jurkat, Hut102 and MOT cells were obtained from Peking Union Medical College (PUMC, Beijing, China). Hut102 is a HTLV-1-infected cell line and MOT is a HTLV-2-infected cell line. All of these cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS (Gibco) in a humidified atmosphere with 5% CO2. gDNA was extracted using a Tiangen Magnetic Blood Genomic DNA Kit (Tiangen Biotech, China).
PcDNA3.1(-)-HTLV-1-RPPH1 plasmid construction and standard curves preparation
To construct the pcDNA3.1(-)-HTLV-1-RPPH1 plasmid, the human RPPH1 gene and the HTLV-1 pol and tax region were amplified using gDNA from Hut102 cells and consecutively cloned into pcDNA3.1(-)-Rluc-Fluc vector (conserved in our lab). The HTLV-1 pol and tax region were separately cut with SpeI endonucleases and cloned into the vector that contains RPPH1 gene (pcDNA3.1(-)-RPPH1) previously generated by replace with the Fluc and Rluc gene cutting with BamHI endonucleases (Fig. 1). The construct was finally verified by direct DNA sequencing (Shenggong, Shanghai, China). The concentration of the standard plasmid was determined by spectrometry at 260 nm. The pcDNA3.1(-)-H1-pol-tax-RPPH1 vector was serially diluted at a range of 5 x 109–1 copies/μL in order to establish a quantitative curve. The standard dilutions that from 5 × 109 copies/μL ~5×102 copies/μL were prepared once and stored at -20℃. The final dilutions (50 copies/μL, 5 copies/μL and 0.2 copies/μL) were prepared immediately before use. All the dilutions and individual samples were run in duplicate.
Selection for specific primers and probes
Primers and probes were designed by Primer 5 to target the conserved sequences of the pol and tax regions of the HTLV-1 and RPPH1 sequences. Primers were screened by efficiency of amplification using SYBR Premix Ex TaqTM II kit (Takara, Dalian, China), and primers with significant dimerization were removed. Probes were further screened using Hyperstart Premix-UNG (Biori, Zhuhai, China) in accordance with the manufacturer’s instructions. Probes with lower count (Ct) values were chosen for further experiments (data not shown). pcDNA3.1(-)-H1-pol-tax-RPPH1 was used as template for primers and probes screening. The primers and probes we finally selected for using were listed in Table 1.
TaqMan real-time quantitative PCR
Real-time qPCRs were conducted using a 7500 ABI (Applied Biosystems). The final concentrations of primers of HTLV-1 pol and RPPH1 were optimized from 0.2μM to 1.0μΜ, while the probes were optimized from 0.2μM to 1.0μM. The annealing temperature for qPCR were optimized from 60℃ to 68℃. The operate concentration of primers and probes, as well as the annealing temperature selected for the following experiments were based on the high efficiency (between 95%~105%) and the lowest Ct values at the same concentration of the template. Finally, the optimized amplification was performed using the following cycling conditions: initial step of 2min/50 °C, denaturation of 5min/95 °C, 40 cycles of 20 sec/95 °C, annealing step of 30 sec/60℃ and elongation step of 20 sec/72 °C. The final volume of the reaction was 25 μL, the optimized primer concentrations for HTLV-1pol and RPPH1 genes were 800 nM, and the optimized probe concentrations for the two genes were 300 nM. The probes were labeled by FAM (HTLV-1 pol)/VIC (RPPH1). All reactions were carried out using Hyperstart Premix-UNG (Biori, Zhuhai, China) in a concentration specified by the manufacturer. Standard curves for HTLV-1 and RPPH1 were accepted when the slopes were between −3.44 and −3.21 (corresponding to PCR efficiencies of between 95% and 105%) and the coefficients of correlation, r2, were >0.98.
Performance Verification for the Established Assay
To evaluate the dynamic range of the HTLV-1 pol and RPPH1, pcDNA3.1(-)-H1-pol-tax-RPPH1 plasmid was serially diluted at the concentrations from 5 × 109 to 0.2 copies/μL and each of them was tested in replicate.
To evaluate the intra- and inter-assay reproducibility, our method quantified three plasmid dilutions (107copies/μL, 103copies/μL, 5copies/μL) 6 times per day for 3 consecutive days. Three different cell concentrations of Hut102 with known proviral loads (0.026 copies /100 cells, 3.025 copies /100 cells, 114.7 copies /100 cells) were prepared and each of them were extracted independently for six times. Their gDNA were carried out in the same TaqMan assay to evaluate the accuracy.
To analyze the specificity of the assay, gDNA extracted from HTLV-1-negative specimens (3 were infected with HBV, 3 were infected with HCV, 3 were infected with treponema pallidum, and 12 were healthy person) and the MOT cell line (infected with HTLV-2) were tested by the assay. Hut102 containing HTLV-1 genome was used as a positive control.
To evaluate the limit of detection (LOD), genomic DNA extracted from a serial dilution of Hut102 cells with Jurkat cells was used as the template, each of which was tested by the duplex qPCR for 20 times. Probit analysis was used to determine the LOD of the assay.
Digital PCR
For QuantiStudio 3D digital PCR analysis, 5 ng of genomic DNA from Hut102 cells and 10ng of pcDNA3.1(-)-H1-pol-tax-RPPH1 plasmid were digested with KpnI (New England Biolabs, Ipswich, MA) was added to digital PCR master mix v2 (Thermo Fisher Scientific, Waltham, MA) with a final concentration of 500 nM forward and reverse primers, and 150 nM of FAM and VIC-labeled probes. The PCR mixture was loaded on the digital PCR chip, and PCR was performed with a LifePro thermal cycler (Bio-Rad) using primers and probes for HTLV-1 and RPPH1 described above and detected with a QX200 droplet reader (Bio-Rad).
Clinical samples
A total of 85 samples from blood donors were included in this study. All of these samples were confirmed as LIA-positive or indeterminate by INNO-LIA HTLV I/II score kit (including 30 HTLV-1-positive, 6 untypeable and 49 indeterminate samples) and simultaneously tested by WB (MP HTLV Blot 2.4, MP Biomedicals, Singapore) and qPCR. DNA was extracted from the whole blood of each blood sample described as above and HTLV-1 proviral DNA was detected and quantified. The normalized HTLV-1 PVL was calculated as the ratio of (HTLV-1 DNA average copy number/RPPH1 average copy number) × 2 × 100 leukocytes, expressed as the number of HTLV-1 copies per 100 cells.