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The Concept of Making On-Chip Thermal Cycler for RT-PCR Using Conjugate Heat Transfer in Diverging Microchannel

ABSTRACT: Covid-19 is pandemic to which the world is fighting. Various precautionary measures are being imposed all over the world which is affecting the routine life of an individual and also the economy worldwide. Although, a definite vaccine is still not known to medical science but they are able to distinguish Covid-19 from the other types of flu. Presently this is being done by detecting the SARS-CoV-2 virus using RT-PCR technique as recommended by the World Health Organization (WHO) (WHO, Geneva, 2020). Reverse Transcription Polymerase Chain Reaction (RT-PCR) is a nucleic acid amplification test that converts the RNA into DNA and subsequently amplifies the specific DNA targets. This method was already being employed to detect the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (Emery et al. in Emerg Infect Dis 10(2):311, 2004). The entire process of DNA amplification takes place in three steps: denaturation, annealing, and extension for which the sample is required to be maintained at constant temperatures of 95, 55 and 72 °C, respectively. This article introduces the technology to achieve a constant temperature which can be tweaked to develop on-chip RT-PCR.

SUBMITTER: Duryodhan V

PROVIDER: S-EPMC7247283 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Project description:BackgroundMany 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 findingsIn 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/significanceWe 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.

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The Concept of Making On-Chip Thermal Cycler for RT-PCR Using Conjugate Heat Transfer in Diverging Microchannel

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