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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

PCR amplification of Neisseria gonorrhoeae porA pseudogene from clinical samples.Lane 1: ladder 50/100/150/200/300/500/800/1500 bp. Lanes 2 to 4: NTC and two positive samples performed with commercial cycler. Lane 5 to 7: NTC and two positive samples performed with TTC.
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pone.0131701.g006: PCR amplification of Neisseria gonorrhoeae porA pseudogene from clinical samples.Lane 1: ladder 50/100/150/200/300/500/800/1500 bp. Lanes 2 to 4: NTC and two positive samples performed with commercial cycler. Lane 5 to 7: NTC and two positive samples performed with TTC.

Mentions: To demonstrate its practical value, we used the TTC to amplify genomic DNA extracted from clinical samples. PCR reactions targeting 132 bp Neisseria gonorrhoeae porA pseudogene using the SsoAdvanced SYBR Green Supermix (Bio-Rad) were tested. The gel electrophoresis data from commercial and TTC reactions are shown in Fig 6. TTC was able to amplify clinical sample-derived bacterial DNA rapidly in 25 min while the commercial protocol took 67 min.


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)

PCR amplification of Neisseria gonorrhoeae porA pseudogene from clinical samples.Lane 1: ladder 50/100/150/200/300/500/800/1500 bp. Lanes 2 to 4: NTC and two positive samples performed with commercial cycler. Lane 5 to 7: NTC and two positive samples performed with TTC.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131701.g006: PCR amplification of Neisseria gonorrhoeae porA pseudogene from clinical samples.Lane 1: ladder 50/100/150/200/300/500/800/1500 bp. Lanes 2 to 4: NTC and two positive samples performed with commercial cycler. Lane 5 to 7: NTC and two positive samples performed with TTC.
Mentions: To demonstrate its practical value, we used the TTC to amplify genomic DNA extracted from clinical samples. PCR reactions targeting 132 bp Neisseria gonorrhoeae porA pseudogene using the SsoAdvanced SYBR Green Supermix (Bio-Rad) were tested. The gel electrophoresis data from commercial and TTC reactions are shown in Fig 6. TTC was able to amplify clinical sample-derived bacterial DNA rapidly in 25 min while the commercial protocol took 67 min.

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