Fig 1.
Setup of the low-cost and rapid TTC.
Major components include three 24-oz thermoses and a pan-and-tilt servo set to control the up, down, and rotational motion to shuttle PCR vessels in and out of the thermoses. Also included are the battery pack, the Arduino electronic controller, and a breadboard. To reduce costs, the pan-and-tilt setup is constructed using a soup can for a fixed height, a wood stick, and a PCR tube holder made with metal wire.
Table 1.
Target, primer sequences, target sizes, and components of PCR.
Fig 2.
(a) Fluorescent intensity of capillary tubes before after PCR. The two tubes on the right are capillary tubes after TTC-PCR [30s/40x(4s/6s) in under 7.5 min]. (b) Gel electrophoresis data after rapid TTC-PCR. Lane 1: ladder. Lanes 2 and 3: duplicate samples that had PCR cycles of 30s/40x(4s/6s) that took under 7.5 min to complete. Lane 4: ladder. Lanes 5 and 6: duplicate samples that had PCR cycles of 30s/40x(2s/4s) that took 5 min to complete. Lane 7: ladder. Lanes 8 and 9: NTC. Lane 10: ladder.
Fig 3.
Eight identical multiplexed PCR reactions performed with TTC.
Lane 1: ladder. Lanes 2 and 3: amplicons produced by the commercial thermal cycler (run time of 84 min). Lane 4: ladder. Lanes 5 to 12: amplicons produced by the TTC using a 180s/35x(15s/2.5s/20s) protocol (run time of 28 min). Lane 13: ladder.
Fig 4.
PCR amplification of Chlamydia trachomatis DNA template from clinical samples.
Lane 1: ladder. Lanes 2 to 5: templates from 1 to 1000X diluted samples amplified for 40 cycles (under 12 min to complete the PCR). Lane 6: ladder. Lanes 7 to 10 contain the same samples as Lanes 2–5 but were only amplified for 35 PCR cycles (10.5 min).
Fig 5.
(a) Gel data for Ebola RNA after RT-PCR. Lane 1: commercial PCR 300s/30s/45x(30s/10s/30s), run time of 69 min. Lanes 2 and 3: TTC run with glass capillary tubes 300s/10s/50x(9s/21s), total run time of 31.9 min. Lanes 4 and 5: TTC run with glass capillary tubes 300s/10s/45x(12s/25s), total run time of 34.5 min. Lane 6: TTC run with plastic tubes 300s/10s/45x(20s/30s), total run time of 44.2 min. Lane 7: ladder. The gel data show that the amplification produced the correct product, and the yield is similar to commercial PCR runs. (b) Real-time RT-PCR plot from the commercial run. The Cq of 36.8 suggests that the TTC can amplify very small amounts of RNA.
Fig 6.
(a) Fluorescent signal from reactions using plastic tubes. The PCR tubes that had undergone 45 cycles of TTC RT-PCR amplification were visualized using a cell phone camera. The top two tubes are HIV positive samples, while the bottom two tubes are the same mix that did not go through the RT-PCR process. (b) Gel electrophoresis data of HIV RNA amplicons after RT-PCR using thin-walled polypropylene tubes. Lane 1: ladder. Lanes 2 and 3: commercial RT-PCR (50 cycles in 83 min). Lanes 4 and 5: TTC 330s/10s/45x(15s/30s) (41 min). Lanes 6 and 7: TTC 330s/20s/45x(15s/30s) (41.3 min). (c) Real-time PCR data from the commercial thermal cycler (Cq = 34.8).
Fig 7.
Gel electrophoresis data of HIV RNA amplification in glass capillary tubes.
Lane 1: ladder. Lanes 2 and 3: commercial run at 300s/30s/45x(10s/30s) in 74 min. Lane 4: ladder. Lanes 5 and 6: 300s/10s/45x(9s/21s) in 28.5 min. Lanes 7 and 8: 300s/10s/40x(9s/16s) in 24.8 min. Lanes 9 and 10: 300s/10s/45x(9s/11s) in 22.1 min. Lanes 11 and 12: 300s/10s/45x(5s/10s) in 17.3 min. Lane 13: ladder. Although the amount of amplicons generated was reduced when shorter protocols were used, a reasonable amount of amplicons was still produced.
Fig 8.
(a) Gel electrophoresis data of the RT-PCR amplification of DENV-1 RNA at different number of PCR cycles. The gel data show that the signal grew exponentially starting between cycles 30 to 35. (b) Real-time plot of TTC vs. commercial cycler using the same PCR mix from (a). The time to complete the specific cycle was plotted against the fluorescence of the PCR mix post-PCR. It took 1068 and 2424 s for TTC and commercial thermal cycler to reach the Cq level, respectively.