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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

Effect of using oil as an insulation to keep water above 90°C for denaturation step during PCR.The water temperature inside the thermos drops significantly faster if oil is not used to insulate the water. Oil can maintain the denaturation and annealing/extension baths’ water temperature very well for over 1 hr. Because our typical 40-cycle reactions does not last longer than 30 min; PCR efficiency carried out by the TTC does not drop significantly even near the end of reaction when exponential growth is having its most effect.
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pone.0131701.g002: Effect of using oil as an insulation to keep water above 90°C for denaturation step during PCR.The water temperature inside the thermos drops significantly faster if oil is not used to insulate the water. Oil can maintain the denaturation and annealing/extension baths’ water temperature very well for over 1 hr. Because our typical 40-cycle reactions does not last longer than 30 min; PCR efficiency carried out by the TTC does not drop significantly even near the end of reaction when exponential growth is having its most effect.

Mentions: We used vacuum-insulated stainless steel food-jars and an oil layer to achieve a low-cost method to passively, but effectively, maintain the temperature of the water baths for PCR. These COTS Thermos food-jars can maintain water temperature effectively only if their caps are tightly closed to prevent heat loss. To perform water bath-based PCR, the caps have to be removed to allow partial immersion of the tubes which will lead to a rather fast temperature drop during the reaction if only water is used. By adding a layer of oil on top of the heated water, we were able to maintain the water temperature in the thermos extremely well (over 90°C for 1 hr) for hot-starting the polymerase and denaturing the target DNA (Fig 2). In our 16-oz thermos (~473 mL max. capacity), as little as 11 mL of oil can sufficiently cover the water surface and build a layer of insulation. Starting at an initial temperature of 95.7°C, the temperature dropped only 4.7°C to 91.0°C in 60 min. Without the oil, the water temperature dropped over 37°C in the same time span. For the annealing and extension steps, which usually take place between 50 and 75°C, the oil and the thermoses help to maintain the temperature bath even better because of the lower temperature difference between the ambient environment and the water inside the annealing/extension bath. For example, in Fig 2 the temperature dropped only 2.7°C in an hour (from 60.5 to 57.8°C) when the oil layer was present. However, the temperature dropped 11.8°C (from 60.0 to 48.2°C) without the oil layer. It is important to note that since we carry out our standard 40-cycler reactions in the 13 to 30 min. time frame, the temperature drop of less than 2.7°C within the denaturation thermos is insignificant. Our data (not shown) also indicates that both cooking oil and mineral oil provide very effective insulation and the difference is negligible, so the use of cooking oil is recommended due to its lower price.


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)

Effect of using oil as an insulation to keep water above 90°C for denaturation step during PCR.The water temperature inside the thermos drops significantly faster if oil is not used to insulate the water. Oil can maintain the denaturation and annealing/extension baths’ water temperature very well for over 1 hr. Because our typical 40-cycle reactions does not last longer than 30 min; PCR efficiency carried out by the TTC does not drop significantly even near the end of reaction when exponential growth is having its most effect.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131701.g002: Effect of using oil as an insulation to keep water above 90°C for denaturation step during PCR.The water temperature inside the thermos drops significantly faster if oil is not used to insulate the water. Oil can maintain the denaturation and annealing/extension baths’ water temperature very well for over 1 hr. Because our typical 40-cycle reactions does not last longer than 30 min; PCR efficiency carried out by the TTC does not drop significantly even near the end of reaction when exponential growth is having its most effect.
Mentions: We used vacuum-insulated stainless steel food-jars and an oil layer to achieve a low-cost method to passively, but effectively, maintain the temperature of the water baths for PCR. These COTS Thermos food-jars can maintain water temperature effectively only if their caps are tightly closed to prevent heat loss. To perform water bath-based PCR, the caps have to be removed to allow partial immersion of the tubes which will lead to a rather fast temperature drop during the reaction if only water is used. By adding a layer of oil on top of the heated water, we were able to maintain the water temperature in the thermos extremely well (over 90°C for 1 hr) for hot-starting the polymerase and denaturing the target DNA (Fig 2). In our 16-oz thermos (~473 mL max. capacity), as little as 11 mL of oil can sufficiently cover the water surface and build a layer of insulation. Starting at an initial temperature of 95.7°C, the temperature dropped only 4.7°C to 91.0°C in 60 min. Without the oil, the water temperature dropped over 37°C in the same time span. For the annealing and extension steps, which usually take place between 50 and 75°C, the oil and the thermoses help to maintain the temperature bath even better because of the lower temperature difference between the ambient environment and the water inside the annealing/extension bath. For example, in Fig 2 the temperature dropped only 2.7°C in an hour (from 60.5 to 57.8°C) when the oil layer was present. However, the temperature dropped 11.8°C (from 60.0 to 48.2°C) without the oil layer. It is important to note that since we carry out our standard 40-cycler reactions in the 13 to 30 min. time frame, the temperature drop of less than 2.7°C within the denaturation thermos is insignificant. Our data (not shown) also indicates that both cooking oil and mineral oil provide very effective insulation and the difference is negligible, so the use of cooking oil is recommended due to its lower price.

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