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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

Multiplexed PCR reactions performed with TTC.Lane 1: Ladder: 50/100/150/200/300/500/800/1500 bp. Lane 2: amplicons produced by the commercial thermal cycler (30 cycles in 75 min). Lane 3: amplicons produced by the TTC (40 cycles in 28 min).
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pone.0131701.g005: Multiplexed PCR reactions performed with TTC.Lane 1: Ladder: 50/100/150/200/300/500/800/1500 bp. Lane 2: amplicons produced by the commercial thermal cycler (30 cycles in 75 min). Lane 3: amplicons produced by the TTC (40 cycles in 28 min).

Mentions: Using the Isohelix DQC kit, we ran the PCR using three thermoses and used gel electrophoresis to confirm that multiple targets could be amplified. The gel photo in Fig 5 shows that the TTC can produce multiplexed amplicons with the correct sizes. While additional cycles were needed to produce a similar amount of amplicons (as evident by gel band intensity), the 28 min reaction time needed by the TTC to perform 40 cycles is much shorter than the protocol performed with commercial thermal cycler (75 min for 30 cycles). We are confident that with some reaction optimization, such as using a longer incubation time at annealing/extension temperatures, can make each PCR cycle as efficient as those achieved by commercial units.


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)

Multiplexed PCR reactions performed with TTC.Lane 1: Ladder: 50/100/150/200/300/500/800/1500 bp. Lane 2: amplicons produced by the commercial thermal cycler (30 cycles in 75 min). Lane 3: amplicons produced by the TTC (40 cycles in 28 min).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131701.g005: Multiplexed PCR reactions performed with TTC.Lane 1: Ladder: 50/100/150/200/300/500/800/1500 bp. Lane 2: amplicons produced by the commercial thermal cycler (30 cycles in 75 min). Lane 3: amplicons produced by the TTC (40 cycles in 28 min).
Mentions: Using the Isohelix DQC kit, we ran the PCR using three thermoses and used gel electrophoresis to confirm that multiple targets could be amplified. The gel photo in Fig 5 shows that the TTC can produce multiplexed amplicons with the correct sizes. While additional cycles were needed to produce a similar amount of amplicons (as evident by gel band intensity), the 28 min reaction time needed by the TTC to perform 40 cycles is much shorter than the protocol performed with commercial thermal cycler (75 min for 30 cycles). We are confident that with some reaction optimization, such as using a longer incubation time at annealing/extension temperatures, can make each PCR cycle as efficient as those achieved by commercial units.

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