Low-Drift-Rate External Cavity Diode Laser

We present the design, construction, and simulation of a simple, low-cost external cavity diode laser with a measured free-running frequency drift rate of 1.4(1)~MHz/h at 852 nm. This performance is achieved via a compact, nearly monolithic aluminum structure to minimize temperature gradients across the laser cavity. We present thermal finite element method simulations which quantify the effects of temperature gradients, and suggest that the drift rate is likely limited by laser-diode aging

A Rapid and Low-Cost PCR Thermal Cycler for Infectious Disease Diagnostics - 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.</p

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.</p

A Rapid and Low-Cost PCR Thermal Cycler for Infectious Disease Diagnostics

<div><p>The ability to make rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a great step forward in global health. Many molecular diagnostic assays are developed based on using thermal cyclers to carry out polymerase chain reaction (PCR) and reverse-transcription PCR for DNA and RNA amplification and detection, respectively. Unfortunately, most commercial thermal cyclers are expensive and need continuous electrical power supply, so they are not suitable for uses in low-resource settings. We have previously reported a low-cost and simple approach to amplify DNA using vacuum insulated stainless steel thermoses food cans, which we have named it thermos thermal cycler or TTC. Here, we describe the use of an improved set up to enable the detection of viral RNA targets by reverse-transcription PCR (RT-PCR), thus expanding the TTC’s ability to identify highly infectious, RNA virus-based diseases in low resource settings. The TTC was successful in demonstrating high-speed and sensitive detection of DNA or RNA targets of sexually transmitted diseases, HIV/AIDS, Ebola hemorrhagic fever, and dengue fever. Our innovative TTC costs less than $200 to build and has a capacity of at least eight tubes. In terms of speed, the TTC’s performance exceeded that of commercial thermal cyclers tested. When coupled with low-cost endpoint detection technologies such as nucleic acid lateral-flow assay or a cell-phone-based fluorescence detector, the TTC will increase the availability of on-site molecular diagnostics in low-resource settings.</p></div

Target, primer sequences, target sizes, and components of PCR.

<p>Target, primer sequences, target sizes, and components of PCR.</p

Gel electrophoresis data of HIV RNA amplification in glass capillary tubes.

<p>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.</p

A Rapid and Low-Cost PCR Thermal Cycler for Infectious Disease Diagnostics - Fig 2

<p><b>(a) Fluorescent intensity of capillary tubes before after PCR</b>. The two tubes on the right are capillary tubes after TTC-PCR [30s/40x(4s/6s) in under 7.5 min]. <b>(b) Gel electrophoresis data after rapid TTC-PCR</b>. 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.</p

Eight identical multiplexed PCR reactions performed with TTC.

<p>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.</p

PCR amplification of <i>Chlamydia trachomatis</i> DNA template from clinical samples.

<p>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).</p

MOESM1 of miR-133a function in the pathogenesis of dedifferentiated liposarcoma

Additional file 1: Figure S1. DDLPS cells reconstituted with miR-133a do not show differences in migration. a Scratch wound assay was performed in DDLPS cell line 246 transduced with lenti-miR-133a (top) or control (bottom). The initial scratch wound mask was created immediately after wound creation and is shown in blue. Phase contrast-images were taken as cells (yellow) migrate in to the wound region over time. b Relative wound density was calculated by Incucyte Scratch Wound Cell Migration Software