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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

Temperature plot of reagent temperature inside a glass capillary tube measured using a thermocouple.(A) The plot recorded 2 min of hot-start step, followed by 40 cycles of moving the tubes between the denaturation (10 s) and annealing/extension (20 s) water baths. The temperature ripple observed is insignificant. (B) The expanded view of the temperature plot that covers the hot-start step and the first 10 cycles. A rough measurement shows that the material inside the capillary tube can reach denaturing temperature in 6 s, and annealing/extension temperature in 10 s.
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pone.0131701.g003: Temperature plot of reagent temperature inside a glass capillary tube measured using a thermocouple.(A) The plot recorded 2 min of hot-start step, followed by 40 cycles of moving the tubes between the denaturation (10 s) and annealing/extension (20 s) water baths. The temperature ripple observed is insignificant. (B) The expanded view of the temperature plot that covers the hot-start step and the first 10 cycles. A rough measurement shows that the material inside the capillary tube can reach denaturing temperature in 6 s, and annealing/extension temperature in 10 s.

Mentions: Fig 3 shows the temperature readings from the thermal probe inside a water-filled glass capillary tube during a 40-cycle PCR run. The plot recorded the 2 min hot-start process and the incubation of the tubes in the denaturation water bath for 7 s and annealing/extension water bath for 13 s. The water temperature in the two baths was also monitored throughout the reaction. The temperature ripple observed is insignificant. The expanded view from the hot-start and the first 10 PCR cycles were also included.


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)

Temperature plot of reagent temperature inside a glass capillary tube measured using a thermocouple.(A) The plot recorded 2 min of hot-start step, followed by 40 cycles of moving the tubes between the denaturation (10 s) and annealing/extension (20 s) water baths. The temperature ripple observed is insignificant. (B) The expanded view of the temperature plot that covers the hot-start step and the first 10 cycles. A rough measurement shows that the material inside the capillary tube can reach denaturing temperature in 6 s, and annealing/extension temperature in 10 s.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131701.g003: Temperature plot of reagent temperature inside a glass capillary tube measured using a thermocouple.(A) The plot recorded 2 min of hot-start step, followed by 40 cycles of moving the tubes between the denaturation (10 s) and annealing/extension (20 s) water baths. The temperature ripple observed is insignificant. (B) The expanded view of the temperature plot that covers the hot-start step and the first 10 cycles. A rough measurement shows that the material inside the capillary tube can reach denaturing temperature in 6 s, and annealing/extension temperature in 10 s.
Mentions: Fig 3 shows the temperature readings from the thermal probe inside a water-filled glass capillary tube during a 40-cycle PCR run. The plot recorded the 2 min hot-start process and the incubation of the tubes in the denaturation water bath for 7 s and annealing/extension water bath for 13 s. The water temperature in the two baths was also monitored throughout the reaction. The temperature ripple observed is insignificant. The expanded view from the hot-start and the first 10 PCR cycles were also included.

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