Molecular Amplification Is More Than Just "PCR"—Here Comes Isothermal Amplification "MIRA"!

Molecular diagnostic technologies today are evolving at an unprecedented pace, with significant advancements over the past two years. Among them, isothermal amplification technologies have gained more attention due to their ability to perform amplification without the need for specialized equipment. Several well-known isothermal amplification methods exist, such as LAMP, RPA, and RAA. However, today I want to introduce a newer isothermal amplification technology—MIRA.

To ensure everyone has a consistent understanding of the key concepts, here are some term explanations:

- Recombinase Polymerase Amplification (RPA): This is a recombinase-mediated amplification technology.

- Recombinase-Aided Amplification (RAA): A similar recombinase-mediated amplification method.

- Loop-Mediated Isothermal Amplification (LAMP): Another isothermal amplification technique.

- Multienzyme Isothermal Rapid Amplification (MIRA): A new isothermal rapid amplification technology.

Quick Summary and Clarification:

Many might wonder which of these various isothermal amplification methods is more reliable and stable. In reality, every technology has its strengths and limitations. However, the key factors for successful adoption are the ability to scale production reliably, low cost, and robust performance across diverse applications.

I’ll now briefly analyze each technology based on what I know:

LAMP (Loop-Mediated Isothermal Amplification)：

Many in the field know that LAMP originated in Japan, developed by Dr. Notomi in 2000 as a novel gene amplification technique. Currently, the ownership of this technology belongs to the Japanese company Eiken Chemical Co., Ltd.

Advantages:

- High sensitivity (2-5 orders of magnitude higher than traditional PCR methods).

- Short reaction time (reactions can be completed in 30-60 minutes).

- No need for special instruments in clinical use (though a real-time turbidimeter is recommended during kit development).

- Easy to operate (whether detecting DNA or RNA, the process involves mixing the reaction solution, enzymes, and template in a reaction tube, then incubating at about 63°C for 30-60 minutes in a water bath or incubator, with results observable by the naked eye).

Disadvantages:

- High sensitivity can result in aerosol contamination once the tube is opened, making it unsuitable for use outside of a PCR laboratory due to a high risk of false positives.

- Primer design is challenging, making it unsuitable for some diseases, especially those caused by viruses with few common sequences.

- Its application is relatively limited.

RPA and RAA Isothermal Amplification Technologies：

These two methods are largely homologous, with the main difference being the source of the recombinase used.

- RPA’s recombinase comes from the T4 bacteriophage.

- RAA’s recombinase is derived from bacteria or fungi.

Key Points of Working Principle:

1. In the presence of ATP, the recombinase binds to primers, forming a protein-nucleic acid complex, which then searches for matching target DNA.

2. Once the primer finds the matching DNA template, ATP hydrolysis provides the energy for recombinase to leave the primer, and DNA polymerase synthesizes a new DNA strand.

3. At the same time, single-stranded DNA-binding proteins (SSB) bind to the displaced DNA strand to prevent reformation of the double-strand.

Advantages:

- The RPA/RAA reaction is very fast, with results achievable in about 30 minutes.

- Sensitivity can reach 10 copies/μL.

- No need for complex equipment during amplification.

- Further development of probe-based or colloidal gold chromatography methods greatly simplifies result interpretation, making them well-suited for use in resource-limited or economically disadvantaged areas.

Disadvantages:

- RPA is a technology developed by the British company TwistDx, which is now owned by Abbott, resulting in high costs, long supply cycles, and lack of technical support. This makes large-scale application and mass production difficult in China.

- RAA is a purely domestic technology, but it lacks stability in terms of interference resistance and product reagent consistency. Moreover, the manufacturing process for functional enzymes needs improvement. Though the technology has been on the market for five years, it has not yet been widely adopted due to stability and versatility issues.

- Both RPA and RAA require primers that are 30-35 bp in length. Shorter primers affect specificity, sensitivity, and amplification speed, making primer design challenging.

MIRA (Multienzyme Isothermal Rapid Amplification):

MIRA is a novel isothermal amplification technology introduced in mid-2019. It is homologous to RPA and RAA.

Although MIRA shares similarities with RPA and RAA in terms of working principles, there are significant differences in enzyme selection, cultivation, purification, and other aspects of large-scale production. Moreover, the technical team behind MIRA’s developer, AmpFuture, has spent nearly 11 years perfecting the technology. It is now fully equipped to support the development of ready-to-use reagents, custom development, research projects, and other customizable services.

Advantages:

1. The functional proteins in MIRA reagents are sourced from various enzymes, with some enzymes undergoing targeted modifications to enhance the efficiency of the core enzyme system, resulting in a more comprehensive system.

2. Strong interference resistance and stability. The MIRA reagents undergo extensive optimization in terms of cofactor selection and concentration, along with freeze-drying production processes, making them more resistant to interference and more stable.

3. Customization and versatility. Due to complete control over the production of functional proteins and a deep understanding of the technology, MIRA can be adapted to various applications, offering different reagent systems and personalized customization services.

4. Proprietary patents. Since MIRA is 100% domestically developed, there is no risk of patent infringement during product development. This gives MIRA a competitive advantage both in the domestic and international markets.

5. A complete production protocol has been established, allowing for stable large-scale production.

Disadvantages:

1. Market recognition is still low, and more data and references from different fields (such as positive rates in different areas) are needed to gain wider acceptance.

2. Primer design still follows the characteristics of RPA or RAA, requiring longer primer or probe sequences, which may not facilitate a quick switch for current PCR users.