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Lysis solution: 4 mol/L guanidine hydrochloride, 1% NP-40, 50 mmol/L Tris/HCl buffer, 20 mmol/L sodium citrate, pH 8.0; wash buffer 1:20% absolute ethanol, 900 mmol/L GITC, 10 mmol/L Tris/HCl buffer, pH 7.5; wash buffer 2: acetone and absolute ethanol at a ratio of 3:1; elution buffer: TE buffer (10 mmol/L Tris/HCl buffer, 1 mmol/L EDTA, pH 8.0).
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QIAamp MinElute Virus Spin Kit (57704, Qiagen); 2019-nCoV nucleic acid detection kit (Fluorescent RT-RAA) (F02R13, Jiangsu Qitian Gene Biotechnology Co., Ltd.); One Step PrimeScript™ RT-PCR Kit (Perfect Real Time) (RR064A, TAKARA); One-step Digital RT-PCR Mix (Sniper Medical Technology Co., Ltd.).
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Sniper DQ24 Digital PCR System (Sniper Medical Technology Co., Ltd.) and Archimed X6 time-resolved real-time fluorescent quantitative PCR system (Rocgene (Beijing) Technology Co., Ltd.) were used.
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An integrated disposable device consisting of a nucleic acid extraction unit and nucleic acid amplification unit was created. The nucleic acid extraction unit included a syringe with a silicone film, a spring, a sealing ring, microtubes, and solution chambers. The nucleic acid amplification unit consisted of an amplification tube, temperature control module, and fluorescence detection module.
The silicone film was placed in a syringe, and the nucleic acid was absorbed. A syringe needle was positioned at the top of the microtube opening at equal intervals and sealed using a silicone rubber plug. The solution chambers were axially parallel to each other and arranged in a wheeled shape. The solution chambers were connected to a silicone rubber plug to seal the top of the solution chamber, and the microtubes were connected to the other end. The relative rotation between the syringe and solution chambers, along with the upward and downward movement of the syringe needle, facilitated the switching between different liquids. The solution was mixed by moving the push-pull rod up and down. The combination of these processes resulted in the purification and amplification of nucleic acids.
The five solution chambers of the nucleic acid extraction unit were pre-filled with the lysis solution, wash buffer 1, wash buffer 2, elution buffer, and nucleic acid amplification reaction mix. Once the sample was added to the first solution chamber of the integrated disposable device, the nucleic acid extraction was initiated. The syringe needle was positioned at the opening of the microtube linked to the first solution chamber by mechanical rotation and by pushing it down on the syringe, which was connected to the first solution chamber. By moving the push-pull rod up and down, the lysis buffer containing the sample was mixed, and the nucleic acid was absorbed onto the silicone film. After the syringe was emptied, it was drawn up, removed, and connected to the other solution chambers, and the previous up-and-down movements were repeated. Finally, 50 µL of mixed amplification reaction solution in the fifth solution chamber was injected into the amplification tube. Nucleic acid amplification was performed according to a predetermined program wherein the temperature control module provided the appropriate temperature and reaction time, and the fluorescence detection module monitored the fluorescence signals. The resulting data were displayed in a concise format.
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After adding 500 µL deionized water to the first solution chamber of the integrated disposable device, the syringe was connected to the first chamber. Precise solution suction and discharge of the syringe transfer were measured using a weighing method, with set volumes of 20, 30, and 60 µL respectively. Each measurement was tested separately 10 times and the mean value was calculated from the resulting values.
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A temperature of 60 °C and 95 °C was set, and the temperature of a 50 µL mixed amplification reaction solution in the amplification tube was measured using a thermocouple thermometer. The mean value was calculated from ten measured values.
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The reagents were added to the solution chambers or amplification tubes, as shown in Table 1. The openings of the solution chambers were sealed with silicone rubber plugs to complete the integrated disposable device for SARS-CoV-2 nucleic acid extraction and amplification.
Table 1. Reagents used for the integrated disposable SARS-CoV-2 nucleic acid extraction and amplification device
Number of solution chambers
or amplification tubeSolution 1 300 µL of lysis buffer and 250 µL of absolute ethanol 2 800 µL of wash buffer 1 3 1,200 µL of wash buffer 2 4 200 µL of TE Buffer 5 SARS-Cov-2 nucleic acid amplification solution Amplification tube SARS-Cov-2 nucleic acid amplification mix (powder) -
A recombinant lentivirus containing the N gene 28273-29557 sequence of SARS-CoV-2 (Accession Number: NC_045512) was constructed. The sequence was then synthesized and inserted into the plvx-puro plasmid. The correct plasmid clone (plvx-SARS2-N) was selected using restriction enzyme digestion and sequencing. The plvx-SARS2-N and lentiviral backbone plasmids were co-transfected into 293T cells. The culture medium was replaced at 8 h post-transfection. The cell culture supernatants were collected at 48 and 72 h. The cell culture supernatant was centrifuged at 3,000 ×g for 30 min, filtered through a 0.45 µmol/L syringe filter, and centrifuged at 80,000 ×g for 1.5 h. The pellet was resuspended in PBS and residual nucleic acid was removed using BeyoZonase™ Super Nuclease. The recombinant lentivirus was aliquoted and stored at −80 °C.
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Digital RT-PCR of SARS-CoV-2 nucleic acids was performed using a One-step Digital RT-PCR Mix (Sniper Medical Technology Co., Ltd.). For each reaction, master mix (11 μL), enzyme mix (2 μL), forward and reverse primers (1 μL each, 500 nmol/L), and probe (0.5 μL, 250 nmol/L) were added to 6.5 μL RNA. The reaction conditions were as follows: 50 °C for 5 min and 95 °C for 3 min for reverse transcription, followed by 40 cycles of denaturation at 95 °C for 30 s and annealing/extension at 60 °C for 45 s. The copy number of SARS-CoV-2 nucleic acids was calculated and used as a quantitative standard for the absolute quantitative RT-PCR detection of SARS-CoV-2 nucleic acids.
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After adding 600 µL of the recombinant lentivirus carrying the SARS-COV-2 N gene into the first solution chamber of the integrated disposable device, nucleic acid extraction was done as described above as follows: 1) The syringe was connected to the first solution chamber and the lysis buffer containing the sample was mixed and absorbed onto the silicone film by moving a push-pull rod up and down 40 times; 2) The syringe was connected to the second solution chamber and the push-pull rod was moved up and down 10 times; 3) The syringe was connected to the third solution chamber and the push-pull rod was moved up and down 10 times; and 4) The syringe was connected to the fourth solution chamber and the push-pull rod was moved up and down 10 times to obtain the SARS-CoV-2 nucleic acid.
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Nucleic acids were extracted from an equivalent amount of recombinant lentivirus carrying the SARS-COV-2 N gene based on the manufacturer’s instructions (QIAamp MinElute Virus Spin Kit, Qiagen).
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Fluorescent RT-qPCR amplification of the SARS-CoV-2 nucleic acid was done using a one-step RT-qPCR reagent (TAKARA). For each reaction, 2× One-Step RT-PCR Buffer III (10 μL), TaKaRa Ex Taq HS (0.4 μL), PrimeScript RT Enzyme Mix II (0.4 μL), forward and reverse primers (0.4 μL each, 200 nmol/L), and probe (0.8 μL, 400 nmol/L) were added to 7.6 μL RNA. The reaction conditions were as follows: 50 °C for 5 min and 95 °C for 3 min for reverse transcription, followed by 40 cycles of denaturation at 95 °C for 30 s and annealing/extension at 60 °C for 45 s. Nucleic acids detected by digital RT-PCR were used to establish a standard curve, and the copy numbers of the nucleic acids extracted by the two methods were calculated.
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The quantified SARS-CoV-2 nucleic acids were diluted to 10,000, 1,000, 500, 250, and 50 copies/mL. Nucleic acid extraction and amplification were performed using an integrated disposable device assembled as described above. Nucleic acids were extracted as previously described. Then, 50 μL of mixed amplification reaction solution in the fifth solution chamber was injected into the amplification tube to dissolve the RAA mix powder. The temperature control module maintained the amplification reaction solution at 42 °C for 600 s and the fluorescence detection module collected measured signals every 10 s. The resulting data were displayed in a concise format. The mean value was calculated from three measurements.
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The structure of the integrated disposable device is shown in Figures 1 and 2.
Figure 1. The schematic view and top view of the integrated disposable device. A) a schematic view of the integrated disposable device; B) a top view of the integrated disposable device. 1: syringe; 2: nucleic acid extraction unit; 3: nucleic acid amplification unit; 4: push-pull rod; 5: silicone film; 6: syringe needle; 7: solution chamber; 8: microtube; 9: silicone rubber plug.
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The solution volumes for syringe suction and discharge were set to 20, 30, and 60 μL. The mean values calculated from the measurements were 19.2, 32.2, and 57.2 μL, respectively. The standard deviation is < 10% (Table 2).
Table 2. The measured values of liquid transfer
Set volume (μL) Measured volume (μL) Mean ± SD (μL) 1 2 3 4 5 6 7 8 9 10 20 22 20 18 17 18 21 19 22 17 18 19.2 ± 1.9 30 30 32 34 34 34 31 30 31 33 33 32.2 ± 1.6 60 55 57 57 62 57 56 57 62 50 59 57.2 ± 3.5 -
The temperature of the amplification reaction mix was set to 60 °C and 95 °C. The mean values calculated from ten measurements were 60.0 °C and 95.1 °C, respectively. The standard deviation is < 1% (Table 3).
Table 3. The measured values of temperature
Set value
(°C)Measured value (°C) Mean ± SD
(°C)1 2 3 4 5 6 7 8 9 10 60 60.0 60.0 60.0 60.0 60.0 60.0 60.0 60.0 60.0 60.0 60.0 ± 0.0 95 95.2 95.3 94.9 95.2 95.0 94.9 95.1 95.0 95.3 94.9 95.1 ± 0.2 -
SARS-CoV-2 nucleic acid was measured at 1.12 × 108 copies/mL by digital RT-PCR (Figure 3) and used as a quantitative standard for absolute quantitative RT-PCR.
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The copy numbers of SARS-CoV-2 nucleic acids from the two nucleic acid extraction methods were determined using absolute quantitative RT-PCR. The nucleic acid extracted by the integrated disposable device was 7.10 × 106 copies/mL, whereas that extracted by the commercial kit was 2.98 × 106 copies/mL (Table 4 and Figure 4). The amount of nucleic acid extracted by the integrated disposable device was higher than that extracted using a commercial kit.
Figure 4. Absolute quantitative RT-PCR detection of SARS-CoV-2 nucleic acid extracted by the integrated disposable device and a commercial kit.
Table 4. Copy number of SARS-CoV-2 nucleic acid extracted by the integrated disposable device and commercial kit
Extraction method Copy number 1 (copies/mL) Copy number 2 (copies/mL) Mean value (copies/mL) Integrated disposable device 7.12 × 106 7.08 × 106 7.10 × 106 Commercial kit 3.28 × 106 2.68 × 106 2.98 × 106 -
The mean times to complete SARS-CoV-2 nucleic acid extraction and amplification were 36 min and 45 s (2,187 s), respectively. Amplification curves were obtained with an initial concentration ranging from 250 to 1 × 104 copies/mL, and two of the three reactions at 50 copies/mL. No curves were observed at the initial concentration of 0 (Figure 5 and Table 5). The lower limit of detection was 250 copies/mL.
Table 5. The result of the absolute quantitative RT-PCR test of SARS-CoV-2 nucleic acid
Number Initial Concentration
(copies/mL)Result† 1 2 3 1 1 × 104 + + + 2 1 × 103 + + + 3 5 × 102 + + + 4 250 + + + 5 50 + - + 6 0 - - - Note. †Each test was repeated three times. Positive results are marked as “+”, and negative results are marked as “-.”
doi: 10.3967/bes2024.070
Development of an Integrated Disposable Device for SARS-CoV-2 Nucleic Acid Extraction and Detection
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Abstract:
Objective To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods We designed, developed, and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection. The precision of the liquid transfer and temperature control was tested. A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR). The entire process, from SARS-CoV-2 nucleic acid extraction to amplification, was evaluated. Results The precision of the syringe transfer volume was 19.2 ± 1.9 μL (set value was 20), 32.2 ± 1.6 (set value was 30), and 57.2 ± 3.5 (set value was 60). Temperature control in the amplification tube was measured at 60.0 ± 0.0 °C (set value was 60) and 95.1 ± 0.2 °C (set value was 95) respectively. SARS-Cov-2 nucleic acid extraction yield through the device was 7.10 × 106 copies/mL, while a commercial kit yielded 2.98 × 106 copies/mL. The mean time to complete the entire assay, from SARS-CoV-2 nucleic acid extraction to amplification detection, was 36 min and 45 s. The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL. Conclusion The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test (POCT). -
Key words:
- An integrated disposable device /
- SARS-Cov-2 /
- Nucleic acid detection
&These authors contributed equally to this work.
注释:1) AUTHOR CONTRIBUTIONS: -
Figure 1. The schematic view and top view of the integrated disposable device. A) a schematic view of the integrated disposable device; B) a top view of the integrated disposable device. 1: syringe; 2: nucleic acid extraction unit; 3: nucleic acid amplification unit; 4: push-pull rod; 5: silicone film; 6: syringe needle; 7: solution chamber; 8: microtube; 9: silicone rubber plug.
Table 1. Reagents used for the integrated disposable SARS-CoV-2 nucleic acid extraction and amplification device
Number of solution chambers
or amplification tubeSolution 1 300 µL of lysis buffer and 250 µL of absolute ethanol 2 800 µL of wash buffer 1 3 1,200 µL of wash buffer 2 4 200 µL of TE Buffer 5 SARS-Cov-2 nucleic acid amplification solution Amplification tube SARS-Cov-2 nucleic acid amplification mix (powder) Table 2. The measured values of liquid transfer
Set volume (μL) Measured volume (μL) Mean ± SD (μL) 1 2 3 4 5 6 7 8 9 10 20 22 20 18 17 18 21 19 22 17 18 19.2 ± 1.9 30 30 32 34 34 34 31 30 31 33 33 32.2 ± 1.6 60 55 57 57 62 57 56 57 62 50 59 57.2 ± 3.5 Table 3. The measured values of temperature
Set value
(°C)Measured value (°C) Mean ± SD
(°C)1 2 3 4 5 6 7 8 9 10 60 60.0 60.0 60.0 60.0 60.0 60.0 60.0 60.0 60.0 60.0 60.0 ± 0.0 95 95.2 95.3 94.9 95.2 95.0 94.9 95.1 95.0 95.3 94.9 95.1 ± 0.2 Table 4. Copy number of SARS-CoV-2 nucleic acid extracted by the integrated disposable device and commercial kit
Extraction method Copy number 1 (copies/mL) Copy number 2 (copies/mL) Mean value (copies/mL) Integrated disposable device 7.12 × 106 7.08 × 106 7.10 × 106 Commercial kit 3.28 × 106 2.68 × 106 2.98 × 106 Table 5. The result of the absolute quantitative RT-PCR test of SARS-CoV-2 nucleic acid
Number Initial Concentration
(copies/mL)Result† 1 2 3 1 1 × 104 + + + 2 1 × 103 + + + 3 5 × 102 + + + 4 250 + + + 5 50 + - + 6 0 - - - Note. †Each test was repeated three times. Positive results are marked as “+”, and negative results are marked as “-.” -
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