Traditional PCR detection reagents are usually provided in three components: reaction mix, enzyme mix, and primer-probe, or as a two-tube “A+B” format where components are mixed before use. Before each test, the operator must calculate quantities, mix the components, and prepare the reagents, which can be complex, error-prone, and requires skilled personnel. Furthermore, because these reagents are mixed right before use, any excess may be wasted, and the preparation process can lead to contamination.

The conventional PCR detection “A+B” format barely meets the requirements of regular testing. However, with the demand surge from mass COVID screening and the expansion into international markets, there is increasing demand in the molecular diagnostics market for a one-tube, ready-to-use RNA pre-mixed reagent (RNA Premix) where reaction mix, enzyme mix, and primers-probes are pre-mixed in one tube.

The RNA Premix avoids the need for end-user preparation, simplifying the operation and reducing user error rates. Moreover, RNA Premix offers high stability, facilitating its international distribution, transport, and storage. However, currently, there are no raw materials on the market that offer a stable and universally applicable RNA premix. Only a very limited number of companies have successfully developed RNA premix products, but these reagents still face accelerated stability issues, are project-specific, and often require compromises in speed, making them non-universal.

In a fully pre-mixed system, all components except for the template (enzymes, magnesium ions, dNTPs, primers-probes) are pre-mixed. During long-term storage, primer-primer and primer-probe interactions can create dimers or secondary structures, leading to non-specific amplification, increasing system complexity, and interfering with the main reaction by consuming necessary components. This can cause fluorescence intensity in amplification curves to decrease after accelerated testing, delay in Ct values, or substantial Ct advancements, significantly raising the probability of false positives. While reducing the quantities of key components may temporarily improve short-term stability, it compromises reagent performance, especially in rapid detection scenarios, making this method impractical.

The main technical bottlenecks in RNA premix development currently include:

1.Insufficient deactivation of functional enzyme activities. Incomplete inhibition of the polymerization and cleavage activities of Taq DNA polymerase, as well as low and room-temperature activity in reverse transcriptase, affects premix stability.

2.Significant enzyme activity loss or inactivation during long-term storage, compromising premix performance.

3.Primer dimerization or secondary structures formed between primers and probes during long-term storage or acceleration testing, complicating system reactions and reducing reagent stability.

Solution Path for HZYMES RNA Premix

To address these challenges, HZYMES has focused on developing a premix reagent, fully optimizing Taq DNA polymerase, heat-activated reverse transcriptase, the reaction system, and consumables, ensuring comprehensive validation and enhancement.

Key Optimizations:

1.Taq DNA Polymerase (Antibody Modification)– Developing a heat-activated Taq DNA polymerase specifically adapted for premix use, ensuring full deactivation of polymerization and cleavage activity, while enabling quick reactivation during reactions.

2.Heat-Activated Reverse Transcriptase (Aptamer Modification)– Advancing the development of heat-activated reverse transcriptase for premix use, ensuring that its activity is fully suppressed at low or room temperature, while improving enzyme stability.

3.Reaction System– Using Design of Experiments (DOE) to optimize the reaction system, reducing secondary structure formation between primers-probes, improving low-concentration curve shape, and maximizing detection rate.

4.Consumables– Inadequate consumables can lead to enzyme inactivation or primer-probe adsorption. HZYMES has conducted comparative testing of leading market consumables to compile a list of premix-compatible consumables.

Additionally, HZYMES has conducted extensive research on early-stage dimer inhibition to ensure long-term reagent stability and superior performance.

Achievements of HZYMES RNA Premix

HZYMES’ RNA premix reagent has successfully overcome technical barriers, achieving stability at 37°C for up to 10 days. It offers broad applicability, compatible with most existing market projects, providing market-leading stability while ensuring high sensitivity and compatibility with rapid protocols. This enables clients to achieve ultra-fast, high-sensitivity, and user-friendly detection reagents.

• RNA Premix Reagent (available in cap-tube and pre-dispensed versions)

Additionally, HZYMES offers custom reagent optimization and CRO services such as targeted primer-probe design and optimization.