From NASBA to RPA: Comparing Key Isothermal Amplification Methods

The field of molecular diagnostics has developed at a fast pace during the last several decades, offering an effective option in numerous cases where pathogens, genetic markers, etc. should be identified. The gold standard of nucleic acids amplification is traditional polymerase chain reaction (PCR). Nevertheless, the accurate thermal cycling technique requires by PCR is limiting its portability and the fast nature in point-of-care (POC) and resource-limited contexts. The isothermal approaches provide a paradigm-altering innovation, that is, the possibility to accomplish amplification of nucleic acids without the requirement of intricate thermal cyclers, using a fixed temperature.

To this end, Nucleic Acid Sequence-Based Amplification (NASBA), Recombinase Polymerase Amplification (RPA) and Helicase-Dependent Amplification (HDA) have become important aspects of the contemporary diagnostics. The techniques also have their mechanisms, benefits, and shortcomings which makes it important to learn which is different and better applicable technique in order to make the best choice depending on the target scenario to be applied in.

The article gives a comparative description of NASBA, RPA, and HDA and their principles, advantages and limitations to help researchers, clinicians and laboratory professionals to make informed decisions.

What Is Isothermal Amplification: Introduction All you want to know about Isothermal Amplification.

An isothermal amplification is the process whereby nucleic acid amplification is done under constant temperature generally between 37 and 65 ^oC. Whereas PCR uses temperature cycling to maintain separation, hybridization and synthesis of target strands, isothermal systems use highly specific enzymes to result in breaking and replicating strands without thermal cycling.

When isothermal amplification is used the principal benefits are:

•           Quicker amplification (usually with the range of 10-30 mins)

•           Reduced complexity of equipment needed

•           The ability to comply with field-based or point of care diagnostic devices

A number of isothermal protocols have been derived, NASBA, RPA, HDA have attracted particular interest, owing to their unique turnaround principles and established use in molecular diagnostics.

Nucleic Acid Sequence Based Amplification (NASBA)

Mechanism of NASBA

Nucleic Acid Sequence-Based Amplification (NASBA) is an isothermal method mostly used as RNA amplification protocol. It is active (efficient) at 41^oC and involves three important enzymes:

1. Reverse Transcriptase- it converts RNA into complementary DNA (cDNA)
2. RNase H- Cleaves the RNA chain of RNA-DNA duplexes
3. T7 RNA Polymerase – The one which produces many copies of RNA using cDNA as a template

This is a cycle process which enables exponential amplification of RNA sequences devoid of thermal cycling.

Advantages Associated with NASBA

• RNA-Specific Amplification: It is very useful to detect an RNA virus such as the HIV, Zika, or SARS-CoV-2.
• Quick Response: The average response time is 90-minutes or less.
• Isothermal Simplicity: It only needs a constant-temperature heat block, thus it is portable.

Having limitations of NASBA

Sensitivity to RNase Contamination: RNases have the ability to digest the RNA target and now give false negative results when not handled properly.

• Complex Enzyme Mix: The three enzymes that have to be used in this product need to be balanced very carefully in order to perform.
• Long Reaction Time than RPA: Despite being faster than PCR, NASBA is by and large slower than other more recent isothermal methods.

Recombinase Polymerase Amplification (RPA)

RPA mechanism

Recombinase Polymerase Amplification (RPA) is an innovation isothermal technology that heightens DNA at ambient temperatures (37 ^oC and 42 ^oC).

The most important ones are:

1. Recombinase-Primer Complex Formation: reacting with primers and leading them to the target DNA, recombinase proteins form a complex.
2. Strand Invasion: Invasion of the primer in the double-stranded DNA is done with the help of the Recombinase.
3. Polymerase Extension: Strand displacing polymerase binds to the primer and extends it and re-synthesizes it.
4. Single-Stranded Binding Proteins (SSBs): Helps to stabilize the unwound DNA to avoid re-annealed.

RPA is highly fast, running the possibility of visualization of detectable amplification in 3 – 20 minutes.

Advantages of the RPA

• Ultra-Fast Amplification: Results within a few minutes which is when urgent diagnostics are required.
• Low Temperature requirement: performs in simple arrangements without advanced incubators.
• Impurities tolerant: RPA is tolerant to the inhibitors that are usually present in biological samples.
• Portability: The ideal fit on field-deployable devices and point-of-care.

Get to know more about Recombinase Polymerase Amplification and how it can change your field of molecular diagnostics.

Restrictions of RPA

• Patents: The technology is proprietary and, therefore, can restrict access by organizations and create extra costs in developing assays.
• Primer Design Sensitivity: The primer should be optimized thoroughly to develop certain specific amplification.
• Reduced Maximum Amplification Output: The RPA probe can generate less amplicons than PCR or NASBA and this depends on RPA conditions.

Helicase-Dependent Amplification ( HDA )

HDA mechanism

Helicase- Dependent Amplification (HDA) is a model of a natural process of DNA replication. It has a helicase enzyme to open up a double-stranded DNA replacing the process of melting of the two strands of DNA used in PCR.

In the response, there is generally:

1.         Helicase: It unwinds the DNA or double strands.

2.         Single-Stranded Binding Proteins (SSBs): bind separated DNA strands.

3.         DNA Polymerase: It goes about the synthesis of the DNA strands with primers.

The reaction of HDA is conducted at 65^oC and can amplify the DNA within 30-90 minutes.

Drugs occur in existed and desired HDA forms.

• Thermal Simple: No need of running denaturation cycles.
• Enhances Targets applicable with DNA: Capable of detecting bacterium, viruses and genetic sequences.
• Relatively Simple Components of Reaction: The reaction has less number of enzymatic steps than NASBA.

Restrictions of HDA

Bounded to DNA targets: HDA, in contrast with NASBA, cannot be used to amplify RNA without a pre-reversed transcriptase step.

Longer reaction time: Certainly, slower than RPA, but faster than the common PCR.

Potential of Reduced Sensitivity: HDA has reduced sensitivity in comparison to RPA and NASBA as it may occasionally display reduced sensitivity especially when the target concentrations are low.

Comparative Description: NASBA, RPA and HAD

Selecting an Appropriate Method to Use in an Isothermal Procedure: All the Factors to Take into Account

The choice of isothermal amplification depends on a number of very essential factors:

1. Target nucleic acid type

•           RNA Viruses: NASBA is specially designed to handle the amplification of RNA, whereby RPA can also allow the working with RNA but with supplementary reverse transcription.

DNA Pathogen/Markers RPA and HDA are best suited in DNA amplification.

2. Time Sensitivity

• Urgent Result Delivery: The time delivery of the results is highly demanded and RPA delivers results in less than 20 minutes.
• Reduced Test Constrained: NASBA and HDA can be acceptable when there is no need to deliver the test results as fast as possible.

3. Field or point-of-Care Deployment

• Equipment shortage: RPA does not need high temperatures and is tough, which makes it very good.
• Laboratory-Based Testing: NASBA and HDA are capable of operating even when using minimal but regulated equipment.

4. Samples Type and Purity

•           Crude Samples: RPA is especially beneficial because it allows impurities in its sample.

•           Cleaner inputs: Cleaner input may be needed by NASBA and HDA to work their best.

5. Price and Availability

• Budget Constraints: HDA and NASBA are usually cheaper because there are less limitations on the licensing laws.
• Proprietary Kits: RPA kits may be costly because of the protection of intellectual property.

Future Prospects in Isothermal amplification

The process of isothermal amplification keeps on improving, and the latest developments strive to increase both the speed, accuracy and usable field application. There are developments, which highlight:

• Multiplexing – the ability to detect many targets or pathogens at a time.
• Integrated Detection systems: This is a combination of amplification and real time visual or electronic reading.
• Incorporation of Detection into CRISPR-Based Systems: the use of CRISPR-Cas systems can enhance both specificity and direct detection after amplification.

RPA has especially attracted considerable attention due to its combination with CRISPR-based diagnostic settings in the form of SHERLOCK and DETECTR, which make new prospects in diagnostic speed, precise pathogen recognition.

Conclusion

Isothermal amplification technology such as NASBA as well as RPA and HDA are changing molecular diagnostics. All the methods have their own strengths:

• NASBA is specific and sensitive to RNA detection but handling has to be precise to deal with enzyme prima donna.
• RPA is top in speed, portability, and field application hence very suitable in time-sensitive and point-of-care diagnostics.
• HDA provides a less complicated series of steps in amplifying DNA based upon natural replication but in general slower.

Knowing the mechanisms and advantages / disadvantages of the different techniques and methods, professionals in the lab industry and those conducting research can be able to make informed choice when choosing the best isothermal method to use as they seek to achieve specific diagnostic objectives.

This is likely to increase as technology sees an even bigger application of these isothermal techniques in the widening accessibility of fast, precise, and off-centered Point-of-care diagnostic solutions across the globe.