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A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings.

Wong G, Wong I, Chan K, Hsieh Y, Wong S - PLoS ONE (2015)

Bottom Line: The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place.The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers.When combined with a rapid nucleic acid detection approach, the thermos thermal cycler (TTC) can enable on-site molecular diagnostics in low-resource settings.

View Article: PubMed Central - PubMed

Affiliation: AI Biosciences, Inc., College Station, Texas, United States of America.

ABSTRACT

Background: Many modern molecular diagnostic assays targeting nucleic acids are typically confined to developed countries or to the national reference laboratories of developing-world countries. The ability to make technologies for the rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a revolutionary step forward in global health. Many molecular assays are also developed based on polymerase chain reactions (PCR), which require thermal cyclers that are relatively heavy (>20 pounds) and need continuous electrical power. The temperature ramping speed of most economical thermal cyclers are relatively slow (2 to 3 °C/s) so a polymerase chain reaction can take 1 to 2 hours. Most of all, these thermal cyclers are still too expensive ($2k to $4k) for low-resource setting uses.

Methodology/principal findings: In this article, we demonstrate the development of a low-cost and rapid water bath based thermal cycler that does not require active temperature control or continuous power supply during PCR. This unit costs $130 to build using commercial off-the-shelf items. The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place. Using an Arduino-based microcontroller, we automate the "archaic" method of hand-transferring PCR tubes between water baths.

Conclusions/significance: We demonstrate that this innovative unit can deliver high speed PCR (17 s per PCR cycle) with the potential to go beyond the 1,522 bp long amplicons tested in this study and can amplify from templates down to at least 20 copies per reaction. The unit also accepts regular PCR tubes and glass capillary tubes. The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers. When combined with a rapid nucleic acid detection approach, the thermos thermal cycler (TTC) can enable on-site molecular diagnostics in low-resource settings.

No MeSH data available.


Related in: MedlinePlus

Setup of the low-cost and rapid TTC.Major components include two 16 oz thermoses, a pan-and-tilt servo set to control the up and 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 cost, the pan-and-tilt setup is constructed using a soup can, a wood stick, and a PCR tube holder made by metal wire. The parts were glued together using removable tape.
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pone.0131701.g001: Setup of the low-cost and rapid TTC.Major components include two 16 oz thermoses, a pan-and-tilt servo set to control the up and 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 cost, the pan-and-tilt setup is constructed using a soup can, a wood stick, and a PCR tube holder made by metal wire. The parts were glued together using removable tape.

Mentions: A key element of the device architecture in our new approach is that it uses Thermos stainless steel vacuum-insulated food jars (to store the water that heats the PCR tubes) and a layer of cooking or mineral oil on top of the water to maintain a stable temperature in the 20 to 30 minute time span that is needed to complete the PCR reaction in a TTC (Fig 1). First, water has a high specific heat capacity, which greatly reduces temperature fluctuations when compared to approaches of using heated air in a thermal cycler (e.g., the LightCycler or the Rotor-Gene Q cycler by Roche Life Science and Qiagen, respectively); and a boiling point very near the DNA denaturation temperature, which makes it a convenient denaturation source requiring negligible control for overheating (only steam will be produced). Water is also readily available and can be added into the TTC only when needed, thus reducing the weight of the device. The oil is the key component which prevents significant heat loss from the surface of the water so that the water in the thermos that performs denaturation has a sufficiently high temperature throughout the cycling duration to carry out the nucleic acid denaturation. Because we are able to maintain sufficient temperatures without active heating or cooling once the water is heated, a stable and continuous power supply is no longer needed during the PCR reactions. To perform two or three-step PCR, two or three thermoses, maintained at denaturation and annealing/extension temperatures, are needed.


A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings.

Wong G, Wong I, Chan K, Hsieh Y, Wong S - PLoS ONE (2015)

Setup of the low-cost and rapid TTC.Major components include two 16 oz thermoses, a pan-and-tilt servo set to control the up and 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 cost, the pan-and-tilt setup is constructed using a soup can, a wood stick, and a PCR tube holder made by metal wire. The parts were glued together using removable tape.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492969&req=5

pone.0131701.g001: Setup of the low-cost and rapid TTC.Major components include two 16 oz thermoses, a pan-and-tilt servo set to control the up and 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 cost, the pan-and-tilt setup is constructed using a soup can, a wood stick, and a PCR tube holder made by metal wire. The parts were glued together using removable tape.
Mentions: A key element of the device architecture in our new approach is that it uses Thermos stainless steel vacuum-insulated food jars (to store the water that heats the PCR tubes) and a layer of cooking or mineral oil on top of the water to maintain a stable temperature in the 20 to 30 minute time span that is needed to complete the PCR reaction in a TTC (Fig 1). First, water has a high specific heat capacity, which greatly reduces temperature fluctuations when compared to approaches of using heated air in a thermal cycler (e.g., the LightCycler or the Rotor-Gene Q cycler by Roche Life Science and Qiagen, respectively); and a boiling point very near the DNA denaturation temperature, which makes it a convenient denaturation source requiring negligible control for overheating (only steam will be produced). Water is also readily available and can be added into the TTC only when needed, thus reducing the weight of the device. The oil is the key component which prevents significant heat loss from the surface of the water so that the water in the thermos that performs denaturation has a sufficiently high temperature throughout the cycling duration to carry out the nucleic acid denaturation. Because we are able to maintain sufficient temperatures without active heating or cooling once the water is heated, a stable and continuous power supply is no longer needed during the PCR reactions. To perform two or three-step PCR, two or three thermoses, maintained at denaturation and annealing/extension temperatures, are needed.

Bottom Line: The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place.The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers.When combined with a rapid nucleic acid detection approach, the thermos thermal cycler (TTC) can enable on-site molecular diagnostics in low-resource settings.

View Article: PubMed Central - PubMed

Affiliation: AI Biosciences, Inc., College Station, Texas, United States of America.

ABSTRACT

Background: Many modern molecular diagnostic assays targeting nucleic acids are typically confined to developed countries or to the national reference laboratories of developing-world countries. The ability to make technologies for the rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a revolutionary step forward in global health. Many molecular assays are also developed based on polymerase chain reactions (PCR), which require thermal cyclers that are relatively heavy (>20 pounds) and need continuous electrical power. The temperature ramping speed of most economical thermal cyclers are relatively slow (2 to 3 °C/s) so a polymerase chain reaction can take 1 to 2 hours. Most of all, these thermal cyclers are still too expensive ($2k to $4k) for low-resource setting uses.

Methodology/principal findings: In this article, we demonstrate the development of a low-cost and rapid water bath based thermal cycler that does not require active temperature control or continuous power supply during PCR. This unit costs $130 to build using commercial off-the-shelf items. The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place. Using an Arduino-based microcontroller, we automate the "archaic" method of hand-transferring PCR tubes between water baths.

Conclusions/significance: We demonstrate that this innovative unit can deliver high speed PCR (17 s per PCR cycle) with the potential to go beyond the 1,522 bp long amplicons tested in this study and can amplify from templates down to at least 20 copies per reaction. The unit also accepts regular PCR tubes and glass capillary tubes. The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers. When combined with a rapid nucleic acid detection approach, the thermos thermal cycler (TTC) can enable on-site molecular diagnostics in low-resource settings.

No MeSH data available.


Related in: MedlinePlus