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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

TTC is able to amplify targets over 1,500 bp long in less than 23 min.Lanes 1 and 6 are amplicons from the commercial run. Lane 2 is amplicon from a TTC run of 12 s / 30 s. Lane 3 is from a run of 12 s / 20 s. Lane 4 is a run of 9 s / 20 s (23 min); Lane 5 is also a run of 9 s / 20 s, but some water got in during the run. Lanes 7 to 9 are amplicons collected from glass capillary tubes with the following protocols 12 s / 30 s, 12 s / 20 s, and 9 s / 20 s, respectively. Lane 10 is ladder: 50/100/150/200/300/500/800/1500 bp.
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pone.0131701.g007: TTC is able to amplify targets over 1,500 bp long in less than 23 min.Lanes 1 and 6 are amplicons from the commercial run. Lane 2 is amplicon from a TTC run of 12 s / 30 s. Lane 3 is from a run of 12 s / 20 s. Lane 4 is a run of 9 s / 20 s (23 min); Lane 5 is also a run of 9 s / 20 s, but some water got in during the run. Lanes 7 to 9 are amplicons collected from glass capillary tubes with the following protocols 12 s / 30 s, 12 s / 20 s, and 9 s / 20 s, respectively. Lane 10 is ladder: 50/100/150/200/300/500/800/1500 bp.

Mentions: Using primers to target the entire 16S rRNA gene of bacteria, the TTC produced similar amounts of target (~1,522 bp) when compared to the commercial cycler and the results were confirmed by gel electrophoresis (Fig 7). Initially, a longer incubation time at the annealing/extension step by TTC was chosen prior to optimization. Lane 1 and 6 are amplicons generated from the commercial thermal cycler (60 min). Lane 2 is amplicon from a TTC reaction with 12 s (denaturation) and 30 s (annealing/extension) steps (total time of 31.1 min). Lane 3 is amplicon from a TTC reaction with 12 s (denaturation) and 20 s (annealing/extension) steps (total time of 24.4 min). Lanes 4 and 5 are TTC reactions with 9 s (denaturation) and 20 s (annealing/extension) steps (total time of 22.4 min). Note that lane 5 produced a weaker band because the tube’s cap popped out during the run and some water/oil got into this particular PCR tube. Lanes 7 to 9 are amplicons produced in glass capillary tubes using the three conditions and reagents as the plastic tubes. Our result demonstrates that using a TTC, a 40-cycle reaction targeting a ~1,522 bp segment of bacterial DNA can be completed in as little as 22.4 min using both plastic and glass capillary tubes.


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)

TTC is able to amplify targets over 1,500 bp long in less than 23 min.Lanes 1 and 6 are amplicons from the commercial run. Lane 2 is amplicon from a TTC run of 12 s / 30 s. Lane 3 is from a run of 12 s / 20 s. Lane 4 is a run of 9 s / 20 s (23 min); Lane 5 is also a run of 9 s / 20 s, but some water got in during the run. Lanes 7 to 9 are amplicons collected from glass capillary tubes with the following protocols 12 s / 30 s, 12 s / 20 s, and 9 s / 20 s, respectively. Lane 10 is ladder: 50/100/150/200/300/500/800/1500 bp.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131701.g007: TTC is able to amplify targets over 1,500 bp long in less than 23 min.Lanes 1 and 6 are amplicons from the commercial run. Lane 2 is amplicon from a TTC run of 12 s / 30 s. Lane 3 is from a run of 12 s / 20 s. Lane 4 is a run of 9 s / 20 s (23 min); Lane 5 is also a run of 9 s / 20 s, but some water got in during the run. Lanes 7 to 9 are amplicons collected from glass capillary tubes with the following protocols 12 s / 30 s, 12 s / 20 s, and 9 s / 20 s, respectively. Lane 10 is ladder: 50/100/150/200/300/500/800/1500 bp.
Mentions: Using primers to target the entire 16S rRNA gene of bacteria, the TTC produced similar amounts of target (~1,522 bp) when compared to the commercial cycler and the results were confirmed by gel electrophoresis (Fig 7). Initially, a longer incubation time at the annealing/extension step by TTC was chosen prior to optimization. Lane 1 and 6 are amplicons generated from the commercial thermal cycler (60 min). Lane 2 is amplicon from a TTC reaction with 12 s (denaturation) and 30 s (annealing/extension) steps (total time of 31.1 min). Lane 3 is amplicon from a TTC reaction with 12 s (denaturation) and 20 s (annealing/extension) steps (total time of 24.4 min). Lanes 4 and 5 are TTC reactions with 9 s (denaturation) and 20 s (annealing/extension) steps (total time of 22.4 min). Note that lane 5 produced a weaker band because the tube’s cap popped out during the run and some water/oil got into this particular PCR tube. Lanes 7 to 9 are amplicons produced in glass capillary tubes using the three conditions and reagents as the plastic tubes. Our result demonstrates that using a TTC, a 40-cycle reaction targeting a ~1,522 bp segment of bacterial DNA can be completed in as little as 22.4 min using both plastic and glass capillary tubes.

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