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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

Photos of the PCR tubes illuminated with blue LED.Fluorescent photos of SYBR-Green based PCR reagents tubes after amplification steps in a commercial cycler and the TTC. The intensity of NTC tube is much lower than the tubes containing template DNA.
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pone.0131701.g004: Photos of the PCR tubes illuminated with blue LED.Fluorescent photos of SYBR-Green based PCR reagents tubes after amplification steps in a commercial cycler and the TTC. The intensity of NTC tube is much lower than the tubes containing template DNA.

Mentions: We first used the TTC to amplify a 139 bp region of gram-positive B. subtilis genomic DNA. Four tubes were placed in a commercial thermal cycler and an identical set was used with the TTC. In each set, one tube is a no-template control (NTC) while the three other tubes contain an identical amount of B. subtilis DNA templates extracted in our laboratory (~1,000 copies/μL). Fig 4 is the photo of the PCR tubes after the reaction which clearly shows that the three samples with extracted B. subtilis template DNA have higher fluorescent signals than the NTC’s. While the commercial thermal cycler protocol took 58 min to complete, the TTC took only 30 min to complete a 40-cycle reaction, resulting in 28 min of time savings.


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)

Photos of the PCR tubes illuminated with blue LED.Fluorescent photos of SYBR-Green based PCR reagents tubes after amplification steps in a commercial cycler and the TTC. The intensity of NTC tube is much lower than the tubes containing template DNA.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131701.g004: Photos of the PCR tubes illuminated with blue LED.Fluorescent photos of SYBR-Green based PCR reagents tubes after amplification steps in a commercial cycler and the TTC. The intensity of NTC tube is much lower than the tubes containing template DNA.
Mentions: We first used the TTC to amplify a 139 bp region of gram-positive B. subtilis genomic DNA. Four tubes were placed in a commercial thermal cycler and an identical set was used with the TTC. In each set, one tube is a no-template control (NTC) while the three other tubes contain an identical amount of B. subtilis DNA templates extracted in our laboratory (~1,000 copies/μL). Fig 4 is the photo of the PCR tubes after the reaction which clearly shows that the three samples with extracted B. subtilis template DNA have higher fluorescent signals than the NTC’s. While the commercial thermal cycler protocol took 58 min to complete, the TTC took only 30 min to complete a 40-cycle reaction, resulting in 28 min of time savings.

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