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

Abstract

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.

Rapid, Affordable and Portable Medium-Throughput Molecular Device for Zika Virus

“…The duration of each commercial real-time RT-PCR run was approximately 57 min. A much shorter protocol was used when the speed of the commercial thermal cycler was compared to a Thermos Thermal Cycler (TTC) developed by the authors4041: 50 °C for 30 sec (cDNA synthesis), 95 °C for 10 sec, and 40 cycles of 95 °C for 5 sec and 60 °C for 10 sec [30 s/10 s/40 × (5 s/10 s)]. The duration of this run was 36 min 47 sec.…”

“…After ZIKV RNA templates extracted by the 3D printer were first amplified by real-time RT-PCR using a commercial thermal cycler, the same templates were amplified by RT-PCR using the TTC4041. The TTC is a practical solution for bringing a low-cost, simple-to-operate, and rapid thermal cycler to underserved and developing populations.…”

“…The TTC is a practical solution for bringing a low-cost, simple-to-operate, and rapid thermal cycler to underserved and developing populations. We previously demonstrated rapid PCR and RT-PCR thermal cycling with the TTC at a rate of 15 to 30 sec per cycle4041. The TTC uses a very simple design that performs PCR amplification based on the “archaic” method of “hand-transferring” reaction tubes through a series of water baths, minimizing the temperature ramping time needed for PCR tubes to reach thermal equilibrium.…”

New pathogens, new tricks: emerging, drug‐resistant fungal pathogens and future prospects for antifungal therapeutics

“…PCRbased diagnostics are often used for fungal identification, 197 but require the use of specialized instrumentation. However, new, rapid, low-cost PCR-based technologies [198][199][200] may improve global access to these diagnostic techniques. New technologies are focusing on whole-genome sequencing for pathogen detection.…”

Diagnostics in Ebola Virus Disease in Resource-Rich and Resource-Limited Settings

“…This assay allows amplification and detection in approximately 35 minutes with excellent sensitivity and specificity, although viral nucleic acid extraction must initially be performed. Other novel technologies, such as portable and low-cost molecular systems [60], handheld NAAT devices [61], and the use of nanoparticles to detect isothermally amplified nucleic acids [62], have been described. Another innovative molecular assay showing promise is the feasibility of providing real-time genome sequencing in an outbreak setting [63].…”