Assessment of Cost-Efficient Thermocycler Prototype for Polymerase Chain Reaction and Loop-Mediated Isothermal Amplification

DNA amplification-based diagnostic is the most accurate method among others, especially during the COVID-19 pandemic. Thus, increasing the global demand for instrumentation and amplification reagents locally, hence increasing import. It is a worrying state in terms of logistics and the future domestic market. An effort for domestic production is a must. Previously a cost-efficient thermocycler prototype using Raspberry Pi and Phyton coding is constructed. Thermocycler prototype flow measurement and heat distribution have previously been tested but never put on a real test for DNA amplification diagnostic. This research aims to compare thermocycler prototypes and commercial for in two types of DNA amplification reactions, polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP). PCR is the most and more common method than LAMP, with the main difference of PCR require thermal cycling and LAMP operate in isothermal conditions. LAMP has a quicker reaction time and operates at a lower temperature. DNA pol with high strand displacement activity is used for LAMP,  in this research Bsm pol is used for LAMP and Taq pol for PCR. Since the prototype thermocycler is designed to be as simple and inexpensive as possible for ease of manufacture and accessibility for every layer of society. Hence, its heat control and stability are not as good as a commercial thermocycler, with huge temperature fluctuation resonance from its set-point. That causes prototype incapability of performing PCR, no DNA band at 250-500 bp range in gel electrophoresis. However, the prototype is capable of performing LAMP, existing <100 bp DNA gradient band in gel electrophoresis. The prototype is also capable of performing LAMP below its protocol temperature and time separately, 62?C and 40 minutes compared to the protocol of 66?C and 60 minutes.

DNA amplification-based diagnostic, Thermocycler prototype, Polymerase Chain Reaction (PCR), and Loop-Mediated Isothermal Amplification (LAMP)

    Indonesia’s reliance on the import of thermocycler, among other components, will neglect the total demand domestically and increase the prices. Both local thermocycler prototype is the first step needed for Indonesia to be self-sufficient in its domestic market, regarding DNA amplification technology. Currently, all thermocyclers are still imported from overseas, although many importers are domestic corporations. Indonesia has not tried to develop its own thermocycler.

However, it has produced its reagent (BioCov-19), yet still imported its components (Tunjung et al., 2020).  Additionally, comprehensive knowledge and skills in developing devices related to molecular detection shall benefit other biotechnology fields in the future such as a mapping of Indonesian biodiversity and natural resources (Berawi, 2019).

Polymerase chain reaction (PCR) is the most common and popular DNA amplification diagnostic method (Mullis and Faloona, 1987). However, there are other alternative methods such as loop-mediated isothermal amplification (LAMP) (Notomi et al., 2000; Nagamine et al., 2002; Tomita et al., 2008). The main difference (Table 1) is the temperature requirement throughout the amplification process, PCR requires thermal cycling (denaturation, annealing, and elongation) and LAMP operates at constant temperature (isothermal). Thus, resulting in different instrumentation requirements, thermal cycling demand a higher specification of thermal control compare to the isothermal process. Other differences are the number of primer sets and DNA pol. PCR only uses one set of primer meanwhile, LAMP could use either 2 or 3 sets of primer. DNA pol for isothermal amplification should have high strand displacement activity in its helicase sub-unit. DNA pol from genus bacillus/bacillus has high strain displacement activity and is commercially used in the isothermal reactions, for example, Bst pol from Bacillus stearothermophilus (Li et al., 2017; Lischer et al., 2020).

Table 1 Comparison of PCR and LAMP

    The thermocycler prototype heat control and stability compare to other prototypes and commercial thermocyclers. Thus, it could not perform a conventional PCR reaction. To tackle this problem the PCR duration (denaturation, annealing, and elongation) could be shortened, decreasing the reaction volume, and direct contact between sample and heating element of thermocycler (without microtube). The coding parameter and addition of better components could also increase its heat control and stability. However, the lack of heat control and stability does not hinder the thermocycler prototype's ability to perform LAMP. Due to its lack of heat control and stability, it becomes a more robust thermocycler compare to thermocycler commercial. It is able to perform LAMP at a lower temperature (62^oC instead of 66^oC) and in a shorter period (40 minutes instead of 60 minutes), separately. Future research needs to develop the prototype on its construction and coding to a more advanced prototype, that is eventually suitable for mass production.

    The authors are grateful for the grant PUPT grant NKB 216/UN2.RST/HKP.05.00/2021 for finding this research. In addition, KL and YW as research supervisors and made the research concept. FA as researcher and writer, and MS as research supervisor.

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