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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

Speed and sensitivity of PCR reactions demonstrated by TTC.PCR reactions to amplify 281 bp of the nuc gene from 2000, 200, 20, or 2 copies of S. aureus genomic DNA. (A) Lanes 1 to 4: PCR products from commercial thermal cycler. Lane 5: ladder. Lanes 6 to 9: PCR products from TTC with thin-walled plastic tubes, using a protocol of 2 min hot-start, followed by 40 cycles of 11 s and 17 s denaturation and annealing/extension. The 40-cycle reactions were completed in 22 min. (B) Lanes 1 to 4: PCR products from commercial thermal cycler. Lane 5: ladder. Lanes 6 to 9: PCR products from TTC with glass capillary tubes, using a protocol of 2 min hot-start, followed by 40 cycles of 7 s and 8 s denaturation and annealing/extension. The reactions were completed in 13 min and 3 s.
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pone.0131701.g008: Speed and sensitivity of PCR reactions demonstrated by TTC.PCR reactions to amplify 281 bp of the nuc gene from 2000, 200, 20, or 2 copies of S. aureus genomic DNA. (A) Lanes 1 to 4: PCR products from commercial thermal cycler. Lane 5: ladder. Lanes 6 to 9: PCR products from TTC with thin-walled plastic tubes, using a protocol of 2 min hot-start, followed by 40 cycles of 11 s and 17 s denaturation and annealing/extension. The 40-cycle reactions were completed in 22 min. (B) Lanes 1 to 4: PCR products from commercial thermal cycler. Lane 5: ladder. Lanes 6 to 9: PCR products from TTC with glass capillary tubes, using a protocol of 2 min hot-start, followed by 40 cycles of 7 s and 8 s denaturation and annealing/extension. The reactions were completed in 13 min and 3 s.

Mentions: To firmly demonstrate that the TTC is as efficient as a commercial thermal cycler in amplifying nucleic acid targets, one needs to show that the TTC can achieve a very low detection limit with single digit copies of template in a single reaction. This means that the amplicons from low copy template reactions can be detected by gel electrophoresis or other means using a reasonable number of cycles (45 cycles and below). To show that the TTC not only is fast, but also extremely efficient, we used the S. aureus nuc gene primers (281 bp) and a fast PCR reaction master mix in thin-walled PCR tubes to amplify the extracted template [44]. The reactions were made with serially diluted template to allow us to prepare each reaction to contain 2, 20, 200 or 2000 copies of S. aureus genome (2 μL of template per reaction). The commercial PCR protocol needed 48 min and 18 s. The TTC condition was 2 min. hot-start at 95°C, 40 cycles of [95°C (11 s) and 60°C (17 s)] for a total of 22 min. The gel electrophoresis data (Fig 8A) shows that in 40 PCR cycles, both the TTC and the commercial thermal cycler were able to amplify enough DNA from a 20 copies/reaction sample to give a visible band in gel.


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)

Speed and sensitivity of PCR reactions demonstrated by TTC.PCR reactions to amplify 281 bp of the nuc gene from 2000, 200, 20, or 2 copies of S. aureus genomic DNA. (A) Lanes 1 to 4: PCR products from commercial thermal cycler. Lane 5: ladder. Lanes 6 to 9: PCR products from TTC with thin-walled plastic tubes, using a protocol of 2 min hot-start, followed by 40 cycles of 11 s and 17 s denaturation and annealing/extension. The 40-cycle reactions were completed in 22 min. (B) Lanes 1 to 4: PCR products from commercial thermal cycler. Lane 5: ladder. Lanes 6 to 9: PCR products from TTC with glass capillary tubes, using a protocol of 2 min hot-start, followed by 40 cycles of 7 s and 8 s denaturation and annealing/extension. The reactions were completed in 13 min and 3 s.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131701.g008: Speed and sensitivity of PCR reactions demonstrated by TTC.PCR reactions to amplify 281 bp of the nuc gene from 2000, 200, 20, or 2 copies of S. aureus genomic DNA. (A) Lanes 1 to 4: PCR products from commercial thermal cycler. Lane 5: ladder. Lanes 6 to 9: PCR products from TTC with thin-walled plastic tubes, using a protocol of 2 min hot-start, followed by 40 cycles of 11 s and 17 s denaturation and annealing/extension. The 40-cycle reactions were completed in 22 min. (B) Lanes 1 to 4: PCR products from commercial thermal cycler. Lane 5: ladder. Lanes 6 to 9: PCR products from TTC with glass capillary tubes, using a protocol of 2 min hot-start, followed by 40 cycles of 7 s and 8 s denaturation and annealing/extension. The reactions were completed in 13 min and 3 s.
Mentions: To firmly demonstrate that the TTC is as efficient as a commercial thermal cycler in amplifying nucleic acid targets, one needs to show that the TTC can achieve a very low detection limit with single digit copies of template in a single reaction. This means that the amplicons from low copy template reactions can be detected by gel electrophoresis or other means using a reasonable number of cycles (45 cycles and below). To show that the TTC not only is fast, but also extremely efficient, we used the S. aureus nuc gene primers (281 bp) and a fast PCR reaction master mix in thin-walled PCR tubes to amplify the extracted template [44]. The reactions were made with serially diluted template to allow us to prepare each reaction to contain 2, 20, 200 or 2000 copies of S. aureus genome (2 μL of template per reaction). The commercial PCR protocol needed 48 min and 18 s. The TTC condition was 2 min. hot-start at 95°C, 40 cycles of [95°C (11 s) and 60°C (17 s)] for a total of 22 min. The gel electrophoresis data (Fig 8A) shows that in 40 PCR cycles, both the TTC and the commercial thermal cycler were able to amplify enough DNA from a 20 copies/reaction sample to give a visible band in gel.

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