|Year : 2021 | Volume
| Issue : 1 | Page : 18-23
Determining the feasibility of constructing a real-time polymerase chain reaction instrument locally: The cost of intellectual property rights and research and development
Saptati Bhattacharjee1, Sanjay Bhattacharya2
1 Department of Bioengineering, University of California, Berkeley, CA, USA
2 Department of Microbiology, Tata Medical Center, Kolkata, West Bengal, India
|Date of Submission||27-Apr-2021|
|Date of Decision||27-May-2021|
|Date of Acceptance||24-May-2021|
|Date of Web Publication||16-Sep-2021|
Dr. Sanjay Bhattacharya
14 Major Arterial Road (E-W), Newtown, Rajarhat, Kolkata - 700 160, West Bengal
Source of Support: None, Conflict of Interest: None
BACKGROUND: Real-time polymerase chain reaction (PCR) machines have advanced the field of microbiology by introducing a robust method for high-throughput detection of nucleic acid targets. Unfortunately, these instruments are quite expensive to purchase and maintain, despite containing hardware parts that are relatively inexpensive.
AIMS AND OBJECTIVES: We believe that it may be possible to mitigate costs in low- and middleincome countries (LMICs) by building such a PCR instrument from assembled parts. However, the major hindrance is the cost of intellectual property rights (IPRs), associated with research and development (R and D). The aim of this project was to understand the hardware costs, IPR royalties, profit margins, and the cost of R and D associated with PCR instrument manufacturing.
MATERIALS AND METHODS: We conducted a review of two PCR machines to examine the differences in their technical and hardware specifications and quantify the cost of assembled parts alone. We also ran a parallel assay using the two machines to understand how the differences in technologies affected assay results.
RESULTS AND CONCLUSION: Based on our analysis, we found that assembling a PCR machine using assembled parts can make these instruments more affordable in LMICs. There is a need to have collaboration and agreement between machine licensing, patent, and copyright holders to facilitate this process in LMICs.
Keywords: Intellectual property rights, low cost, low-middle income countries, real-time polymerase chain reaction, system configuration
|How to cite this article:|
Bhattacharjee S, Bhattacharya S. Determining the feasibility of constructing a real-time polymerase chain reaction instrument locally: The cost of intellectual property rights and research and development. J Acad Clin Microbiol 2021;23:18-23
|How to cite this URL:|
Bhattacharjee S, Bhattacharya S. Determining the feasibility of constructing a real-time polymerase chain reaction instrument locally: The cost of intellectual property rights and research and development. J Acad Clin Microbiol [serial online] 2021 [cited 2021 Oct 27];23:18-23. Available from: https://www.jacmjournal.org/text.asp?2021/23/1/18/326047
| Introduction|| |
Real-time polymerase chain reaction (PCR) allows scientists to track the PCR amplification after each cycle, rather than at the very end.,, Quantitative real-time PCRs, multiplex PCR, melting curve analyses and high-resolution melt curve analysis can be conducted on real-time PCR machines.,, Unfortunately, real-time PCR instruments are currently expensive,,, so this technology is still unavailable to low-resource communities. There are very few high-quality equipment built in India. Access to high-quality equipment built locally in India is limited. However, individual parts can be found locally and are therefore relatively inexpensive. Sourcing locally would also allow for potentially easier maintenance, upkeep, and replacement of the complete machine. This study aims to determine whether such a process would be feasible taking into account quality control, cost of research-development (R and D), intellectual property rights (IPRs) and company profit margins.
| Materials and Methods|| |
The study had four phases:
Phase 1: Literature review
This consisted of a literature review of available materials relevant to the two instruments, the ABI 7500 (Applied Biosystems, Carlsbad, USA) and the Rotor-Gene Q 5-Plex (Qiagen, Hilden, Germany). The technical specifications of each system were compiled and compared to understand the differences between the instruments.
Phase 2: Parallel assay study
A parallel assay was conducted in both machines studied to understand how system differences affect results. The presence of fungi (Aspergillus species) was determined in identical assays run by both the systems.
In this phase, we compared the results of an Aspergillus PCR by qualitative method using SYBR Green chemistry. There were four samples: three unknowns and one negative non-template control. Each sample well contained 10 μL of SYBR Green mix, one microliter of forward primer (unique to Aspergillus genus being tested in the particular sample), one microliter reverse primer (again unique to Aspergillus genus) and six microliters of RNase-free water. The negative non-template control contained only 10 μL of SYBR Green mix and 10 μL of RNase-free water.
After Master Mix preparation, target DNA was added to the sample preparation room. Aspergillus DNA had previously been extracted. Two microliters of each of the species targets was added to the respective wells. This brought the final volume of all sample wells to a total of 20 μL.
The Rotor-Gene Q 5-Plex's reactions were done in reaction tubes, while the ABI 7500 used the necessary strip tubes. The thermal cycling reaction for both assays was as follows: hold at 95°C for 15 min and then alternating cycling between 95°C for 30 s and 58°C for 35 s. The assays were run for 35 cycles.
Phase 3: Study of the internal machine parts
The instruments when opened (during maintenance/repair) were examined for internal hardware (ABI 7500). For the Rotor-Gene, the internal parts were studied using an online video available in YouTube. The parts were identified and catalogued to ascertain whether they would be locally available.
Phase 4: Sourcing of components and cost assessment
Each component was sourced online and the costs were organised to create a cost assessment. A final figure of the base price of an instrument was calculated. This sum was the total cost of the most important hardware parts of each instrument and did not include packaging, extra plastics or metals or assembly costs.
| Results|| |
Phase 1: Literature review
There were various differences between the two PCR systems with regard to their technical specifications. Many of these differences arise due to the differences in optical and thermal cycling technologies, but others were by virtue of design differences in the instrument. This information has been organised and summarised in [Table 1].
|Table 1: Technical specifications of Applied Biosystems 7500 and Rotor-Gene Q 5-Plex|
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Beyond these technical specifications, there were further requirements that differ between the two equipment. [Table 2] and [Table 3] compare the performance and construction of the two PCR systems (Rotor-Gene Q 5-Plex versus ABI 7500) in various categories, including: mechanical parts, software, sample formats, optical systems, materials, electrical parts, and consumables. This information is organised in the form of a comparison matrix on a ranking scale of 0, 0.5 and 1.
|Table 2: Legend for rankings of system requirements of Applied Biosystems 7500 and Rotor-Gene Q 5-Plex|
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|Table 3: Comparing system requirements of Applied Biosystems 7500 and Rotor-Gene Q 5-Plex|
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The fluorescent detection system of the Rotor-Gene Q 5-Plex was ranked higher than that of the ABI 7500 because the photomultiplier tube (PMT) is actually a more sensitive means of detection and can detect low signals as well. In addition, air cycling was ranked higher than the Peltier system for thermal cycling because Peltier blocks may result in non-uniform heating. Although this application may be beneficial in forming thermal gradients, it is more important to have the ability to uniformly heat and cool samples with very low well-to-well variation. This purpose is better achieved using air cycling as compared to Peltier blocks.
Phase 2: Parallel assay in Rotor-Gene and ABI 7500
There are three checkpoints to keep in mind when comparing Ct values of different assays: sample assurance, PCR chemistry and instrumental assurance. In regard to sample assurance, it is important to confirm whether the DNA concentration, extraction date and purity are the same between the samples. In this particular case, since the DNA for both assays were from the same stock, it is unlikely that there would be significant differences in the samples which would contribute to the differences in Ct values. Instrumental assurance, on the other hand, involves checking when the machines were calibrated and ensuring that the instruments are functioning correctly. Both machines used in this assay were calibrated on time. Chemistry used was same, however the reaction volume was different. It is not possible to truly and effectively compare the Ct values of the two assays, especially given that only a single trial was run. However, it is important to note the difference in Ct values [Table 4] for both assays as this demonstrates how drastically instrumentation differences can affect the Ct values.
Phase 3: Internal hardware study
The main components of the ABI 7500 consist of electrical, mechanical and optical parts. As part of its electrical system, the machine contains a switch mode power supply (SMPS), a power amplifier and a Gemini board. In its mechanical parts, the machine has four cooling fans, a heated cover with optical lenses and a Peltier block. The optical system is composed of a tungsten halogen lamp, a charge-coupled device (CCD) camera and a filter assembly consisting of five excitation/emission filters as well as a series of mirrors to reflect and focus the light.
The optical system of the ABI 7500 consists of a tungsten halogen lamp, a filter assembly and a CCD camera for fluorescent detection. A common problem is the collection of dust or carbon on the lamp filament. This interferes with the brightness of the bulb and affects the accuracy of fluorescent detection. The light emitted by the halogen lamp is focused using a series of mirrors in the suspended optical assembly as well as by lenses located in the heated cover. The light also passes through the filter assembly along the way to the samples, allowing only a single wavelength of light through. This excitation wavelength is determined by the type of filter, which in turn is preprogrammed by the software based on the fluorophore of choice. The emitted light from the fluorophore is detected by the CCD camera and is recorded and quantified by the software.
Thermal cycling in the ABI 7500 is achieved by a Peltier block. The Peltier block utilises a principle discovered by Jean Charles Athanase Peltier (thus the name) that converts the flow of a current to either the generation or loss of heat. In other words, a gradient in charge can induce a gradient in temperature. In a Peltier block, two semiconductors of different energy (more specifically, of different Fermi levels) are placed in parallel to each other with a conducting plate in between so that electrons may flow between them. The passing current generates a thermodynamic gradient. The direction of the current determines whether the Peltier block is heating or cooling.
The main components of the Rotor-Gene Q 5-Plex also consist of electrical, mechanical and optical parts. As part of its electrical system, the machine contains a SMPS, a main board, a power board and a sensor board with a thermal fuse. Like the ABI 7500, it also requires a uninterrupted power supply (UPS), which is located externally in our laboratory. In its mechanical parts, the machine has two cooling fans, a centrifugal rotor and air vents. The optical system is composed of an LED light assembly, a filter assembly consisting of five excitation/emission filters and a photomultiplier tube for detection.
The sensor and fuse assembly are a safety mechanism for the instrument. The sensor calculates the speed of the heater fan by detecting the vane of the fan as it passes by the sensor. The speed can then be regulated. The fuse, on the other hand, determines if the chamber temperature exceeds 130°C. If the chamber is too hot, the fuse will blow and will need to be replaced. The blown fuse will switch off the heater's power supply.
The Rotor-Gene Q 5-Plex has a different optical detection and thermal cycling system from the ABI 7500. Instead of a Peltier block, the Rotor-Gene Q 5-Plex uses air cycling to heat and cool PCR samples. Heat is generated from a nickel-chrome element in the lid. The samples spin at 400 rpm in a column of hot air. Then, air vents in the lid and body of the instrument allow cool air generated by an internal fan to push warm air out of the machine through the vents and cool the samples. Temperature sensors in the lid maintain and regulate the temperature within the chamber. This method provides fast and uniform thermal cycling.
The optical system in Rotor-Gene depends on high-energy LEDs (LEDs) instead of a halogen lamp. For the Rotor-Gene Q 5-Plex, there are five different LEDs that encompass a wide variety of wavelengths. In addition, there are five emission filters that are used along with a photomultiplier tube (PMT) to detect fluorescence. The sample tubes spin in the rotor. Every 150 ms, a sample tube will pass the LED assembly and become illuminated. The light is reflected at an angle to be passed through an emission filter before being detected by the PMT.
Phase 4: Sourcing of components and cost assessment
[Table 5] is an estimated cost analysis and comparison of the costs (in Indian Rupees, INR) of the most important components of each instrument (FY 2015–2016). We took the FY 2015–2016 for cost assessment as it represented the mid-year from the time of purchase of the equipment to the current. Furthermore, the initial cost assessment was done around that financial year.
|Table 5: Cost assessment summary for a real-time polymerase chain reaction equipment parts (in Indian Rupees)|
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While some parts are unavailable in India, it is evident that the sum of the costs of the most expensive parts amounts to much less than the total price of the machines. Even accounting for an extra margin of additional parts and labour, the total price of an instrument assembled locally would still be competitively lower than the market price of an imported instrument.
| Discussion|| |
PCR machines are efficient tools in molecular diagnostic laboratories. They are used in a wide range of applications, for example, in diagnostic microbiology, molecular pathology/haematology and basic or translational research. However, these tools of molecular biology are expensive, especially in the context of resource-constrained or resource-limited settings. The high costs of the machines arise because of the research work associated with their development, IPRs and patents, calibration and quality control of equipment, clinical validation and standardisation and, last but not least, business profitability-related reasons. However, the high cost of these machines (which is generally in excess of one million rupees) is beyond the reach of many resource-limited, diagnostic and research laboratories in low- and low-middle income countries. This divide between technology availability and its access, caused by a lack of resources, leads to huge disparities in diagnostic capabilities and patient care. It may be argued that if these technologies could be made available at an affordable price, many more patients and research projects could benefit from the move.
With this background, an attempt was made to understand the technical feasibility of building a PCR machine locally and assessing its cost based on local hardware parts. Two real-time PCR machines, the ABI 7500 and the Rotor-Gene Q 5-Plex, were studied and compared across several metrics as proxies to better understand the process of building a PCR machine from scratch. The methodology used in this technical study included: (1) a literature review of machine manuals available on the internet, (2) a study of internal hardware, (3) a comparison of the results of a parallel assay of qualitative PCRs and (4) a cost analysis of internal hardware components.
The results of this exercise showed that there are significant differences in the internal components of the PCR machines of the two types studied, but these parts could be sourced individually at a much cheaper rate [Table 5]. The principal constraints include (a) IPRs issues, (b) sourcing the individual components of the equipment locally, (c) assembling the machines themselves and (d) validating the machine for diagnostic applications. Both the ABI 7500 and the Rotor-Gene Q 5-Plex are covered by US patents. However, the PCR patent has expired in 2005. Ensuring operational qualifications and performance qualifications of a machine requires time, resources and expertise. This project goes on to show that the development of an assembled PCR machine is conceptually feasible and financially viable, provided that the technical, financial and legal hurdles can be overcome. Scientists and researchers working with PCR technology should have an in-depth understanding of PCR machine components in order to troubleshoot or appreciate nuances of assay variation (Phase 3 of the study highlights this through parallel assay study).
To conclude, technological progress should act as a gateway of opportunity, not only for good business and excellent science, but also to improve the quality of life of the society, in general, and the low-resource settings, in particular.
The authors are grateful to the Tata Social Internship Program for providing the opportunity to pursue this research.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]