Findings consisting of pathogens, genetic markers, or sets of DNA/RNA sequences can be used in the precise and quick identification of diseases and is especially essential in point-of-care (POC) situations. Nucleic acid amplification Polymerase Chain Reaction (PCR) has remained the gold standard in nucleic acid amplification, over decades. Nonetheless, sensitivity to having accurate temperature cycling of the precise requirement and requiring more elaborate equipment compromises en use of PCR within low resource settings and in the field.
Recently, an alternative family of isothermal amplification based Loop-mediated Isothermal Amplification (LAMP) and Recombinase Polymerase Amplification (RPA) have been developed that allowed rapid, cheap, and power-independent molecular diagnosis. Such techniques are being especially disruptive to POC diagnostic tools as it provides a fast, inexpensive and mobile solution where conventional PCR has failed to do so.
This article examines the challenges to the conventional PCR procedure that isothermal amplification procedures have solved, their working principle and how they are core to the following certain generation of POC diagnostics.
Limitations of the Traditional PCR Overview Limitations of the Traditional PCR
The traditional PCR may fail to produce a complete nucleotide sequence Since not all nucleotide sequences can be obtained, the technique is not very useful in tests to detect the presence of viruses generally The technique may produce indefinite lengths of nucleotide Overview The traditional PCR may not result in the acquisition of a complete nucleotide sequence As not all the nucleotide sequences can be obtained, the technique becomes inapplicable New tests to determine the existence of viruses is generally not very useful The technique could yield und
Since its invention in 1980s, practice of PCR has stood out as a pillar in molecular biology. It is ensured by the enhancement of particular sequences of the DNA compositions via a repeating cycle of denaturation, annealing and extending procedures all demanding exact alterations of temperature. Although being very sensitive and specific, PCR has various challenges, especially in decentralized or resource-limited environments.
The Downsides of the Conventional PCR:
On its part, PCR depends on thermal cycler to control the temperature changes accurately, which makes it reliant on the electric power and highly advanced machine.
• Long Turnaround Time: The reaction of standard PCR may require 1-2 hours or longer and it is not suitable to provide rapid testing.
• Composite workflow: PCR usually utilizes skilled staff, several reagents, and high degree of lab conditions.
• Poor Field Applicability: Unreliable electricity supply, controlled conditions and heavy equipment limit the use of PCR in the field settings or in rural places.
These restrictions have been driving the quest towards a more versatile, transportable and high speed alternative- The emergence of isothermal methods of amplification.
What is an Isothermal Amplification?
Isothermal amplification approaches make nucleic acid amplification simple through the axing of the thermal cycling. In difference to PCR, these methods are kept in a fixed temperature, which is usually near to ambient or physiological temperatures. This is introduced as the major difference since amplification can be done with little equipment or even without electricity.
The benefits of Isothermal Amplification:
• No Requirement of Thermal Cycling: Reactions take only one temperature and this considerably reduces the process.
Quick results: The amplification may take between 10-30 min.
• Field-Friendly: The techniques may be done by using simple tools such as a water bath, a portable heater or even the body heat.
• Robustness: It is not sensitive to inhibitors likely to be present in crude samples, so that there is no necessity to subject the sample to a lot of sample preparation before carrying out the test.
Now, we are going to look into two of the most popular isothermal methods of amplification namely LAMP and RPA.
Loop- Mediated Isothermal Amplification (LAMP)
The working of LAMP How LAMP Works
Loop-mediated Isothermal Amplification (LAMP) is a high throughput and highly specific and efficient, isothermal DNA amplification protocol developed by Notomi et al., in the year 2000. The method is used:
• Highly strand displacing DNA polymerase.
• Four to six primers which specifically targeting the target DNA (four to six times more specific).
LAMP The LAMP reaction generates prodigious quantities of DNA in a characteristic loop conformation, which is typically monitored by turbidity, change in colour or by fluorescence.
Some benefits of LAMP
• Speed: The results can be deduced in 15-30 minutes.
• Visual Detection: The visual detection can be performed on an eye level because the amplification can be detected through the colorimetric dyes without employing elaborate readout devices.
• High Sensitivity: Ability of detection levels of low concentrations of target DNA and RNA.
Field Applicability: Has good applicability in crude samples and no need of complex purification of nucleic acids.
The uses of LAMP
• Infectious Disease Detection: It is applied in diagnosing malaria, tuberculosis and COVID-19.
• Food Safety: Diagnosis of food identified pathogens.
Agricultural Testing: Detection of disease and pests in plants.
The efficiency and ease of use of LAMP and the ability to read the results visually makes it an outstanding candidate of POC diagnostic devices in both developed and developing healthcare systems.
RPA (recombinase Polymerase Amplification)
The Way RPA Works
Another isothermal method run at even lower temperatures (i.e. 37-42 o C) has been introduced in the early 2000s: Recombinase Polymerase Amplification (RPA). RPA uses:
• Recombinase proteins which help priming the target DNA.
• DNA synthesis, where the strand displacing polymerases were used.
RPA is able to scale-up ten- to twenty-fold within ten to twenty minutes, and is exquisitely versatile when combined with lateral flow strips, fluorescent reporters, or even straightforward colorimetric outputs.
Positive factors of RPA
• Ultra-Rapid Results Amplification: this can frequently yield results within less than 20 minutes.
• Low Temperature Operation: It can operate under the minimal heat or even body heat.
• Easy To Detect: It is easy to detect as it can be used with a lateral flow device.
• Electricity-Free Potential: Applicable in the relatively inaccessible places, where power may not be readily available.
The following are some of the applications of RPA.
• Diagnostic of Infectious diseases: Quick detection of HIV, Zika virus and SARS-CoV-2.
Veterinary Medicine: Detection of pathogens in Veterinary medicine.
• Water Monitoring: Rapid detection of water waterways contaminants.
Ultra-fast turnaround and low temperatures make RPA outstanding as a solution to field-based, rapid diagnostics.
Comparison Isothermal Amplification and Traditional PCR
| Feature | PCR | LAMP | RPA |
| Temperature Requirements | Thermal cycling (up to 95°C) | Constant (60-65°C) | Constant (37-42°C) |
| Time to Results | 1-2 hours | 15-30 minutes | 10-20 minutes |
| Equipment | Thermal cycler | Simple heat source | Minimal heat source |
| Detection | Fluorescence, gel electrophoresis | Visual (color change, turbidity) | Visual (lateral flow, colorimetric) |
| Field Use | Limited | Highly suitable | Highly suitable |
| Power Dependency | High | Low | Very low |
The comparison explains clearly the similar reasons LAMP and RPA are the core of contemporary POC tools used in diagnostic predominantly in decentralized models.
Real World Impact: Changing the World of Low-Resource Places
The second most notable contribution of isothermal amplification is that it has succeeded in making molecular diagnostics at places that the conventional PCR cannot work. In both rural health clinics and mobile health clinics, as well as emergency outbreak areas, the quick diagnosis is paramount in terms of managing the spread of the disease and prompt treatment.
Case Study LAMP in malaria detection
LAMP-based tests on malaria led to a commendable increase in the capacity to detect low-level parasitemia missed in the conventional microscopy in the sub-Saharan region of Africa. Malaria diagnosis can now take place in field using handheld portable LAMP kits which require only a simple heating block to visualize the color change.
The Case: RPA for Zika Virus
RPA assays were used to achieve rapid and accurate results throughout the Zika outbreaks in Latin America in regions where it was not possible to access conventional laboratories. Low-temperature needs of RPA encouraged health workers to conduct tests with battery driven incubators.
These illustrations highlight the importance of isothermal amplification in facilitating democratization of the molecular diagnostics.
Synergy to the Modern POC Diagnostic Devices
Isothermal amplification tools are used more and more within the new diagnostic modules:
• Lab on Chip Systems: The microfluidic devices integrate sample preparation, amplification and detection on a single, handheld device.
• Lateral Flow Strips: RPA products may be visualized using simple strips of paper, just like pregnancy tests.
• Smartphone-Ready Readouts: Isothermal-based amplifier devices are currently linked with smartphones so that real-time analysis can be read and shared remotely.
The developments are shifting the paradigm of POC diagnostic devices where advanced in-lab testing has come into the hands of patients.
Issues and Courses of Action
Though isothermal amplification has obvious benefits, numerous pitfalls are involved:
• Non-Specific Amplification: High amplification efficiency under naturally designed assay can relatively result in false positive cases.
• Primer Design Complexity: Methods such as LAMP have the problem that these methods need numerous primers and the chance of design failure is more troublesome.
Stability of reagents: Since enzymes and reagents often have limited life span under various environmental conditions, long term stability of reagents and enzymes in the field is crucial.
These challenges are actively discussed by researchers as follows:
• Better algorithms of the primer design.
• Better storage as development of lyophilized (freeze-dried) reagents.
• Combination with CRISPR based sensing platforms in order to improve specificity.
The combination of isothermal amplification and CRISPR diagnostics is a potentially even more promising frontier, with potential still further improvement of testing accuracy, speed, and portability.
Conclusion
Techniques of isothermal amplification such as the LAMP and RPA are fundamentally transforming the molecular diagnostics frontier, and escaping the constraints that typify conventional PCR. They are simple, fast, and flexible devices that can be used in any ILS-point-of-care and field settings, particularly in low-resource areas.
Allowing such electricity-free, fast, and simple-to-operate molecular tests, these techniques dominate the new generation of POC diagnostic tools that are changing the access to healthcare globally.
These techniques are constantly being improved upon and as it continues to merge with the sphere of current portable devices, isothermal will definitely lead the way through diagnostic innovation once more- being the highly sought-after, quick, accurate, universally-available molecular diagnostics that everyone is pursuing.
