New Product Empowerment for Fully Premixed Reagents: Warm-Start Reverse Transcriptase – Fast, Accurate, and Durable - Catalyzing life Smart Manufacturing for the Future-Hzymes Biotechnology

Background

As winter approaches, the incidence of seasonal influenza, pneumonia, and other respiratory diseases increases, leading to a surge in hospital visits. Timely and effective treatment, along with rapid and accurate diagnostic methods, are crucial. Among various testing methods and reagents, the one-step RT-qPCR method (all-in-one RNA premixed reagent) is widely favored for its efficiency, convenience, and sensitivity.

In the one-step reaction, gene-specific primers are theoretically supposed to match with the target for detection and synthesize the first strand of cDNA under the catalysis of reverse transcriptase. However, in practice, non-specific template-primer matching often leads to the production of non-specific cDNA. Non-specific cDNA can cause non-specific amplification within the system, consuming components needed for the reaction and limiting the subsequent main reaction. When the concentration of target nucleic acid is low, competition can lead to poor amplification performance, such as poor amplification curve shape, low fluorescence increase, and delayed Ct values, thereby greatly reducing the sensitivity and specificity of the detection reagent.

Additionally, since the all-in-one RNA premixed reagent mixes enzyme solution, primer probes, and reaction liquid in advance, the enzyme activity can easily decline or become inactive during long-term storage, leading to performance issues with the premixed reagent. The reverse transcriptase (RT) enzyme, as a key component, directly affects the accuracy and sensitivity of one-step nucleic acid detection reagents. Enhancing the specificity and stability of reverse transcriptase is crucial for the development of all-in-one RNA premixed reagents.

In light of this, Hzymes has developed a warm-start reverse transcriptase for one-step RT-qPCR/PCR scenarios and all-in-one RNA premixed reagent development. This enzyme has its activity sealed at low temperatures and activated at working temperatures, avoiding primer-template mismatching and reverse transcription at low temperatures, thereby improving detection specificity and sensitivity. The enzyme is modified with an aptamer method. Hzymes utilizes an aptamer screening platform to prepare a vast library of random oligonucleotide sequences. Based on traditional SELEX aptamer screening technology, it optimizes and improves the process by using high-throughput sequencing instead of traditional cloning sequencing. After several rounds of screening and enrichment, aptamers with high affinity for reverse transcriptase are selected. These high-affinity sequences are then incubated with reverse transcriptase, and the enzyme activity at low and working temperatures is tested to determine the optimal sealing and activation effects. The aptamers are further combined and the enzyme modification process is optimized. Hzymes has developed a warm-start reverse transcriptase that can seal 99% of its activity at 42°C, offering better sealing effect and stronger stability compared to similar products on the market. This ensures that the reaction liquid can continuously and stably obtain reverse transcription products, solving the storage stability problem of premixed reagents. Meanwhile, the product can release more than 95% of its enzyme activity at the working temperature (55°C), ensuring the amplification sensitivity of the reagent.

Figure 1. Hzymes warm-start reverse transcriptase sealing principle diagram (A) and aptamer screening flowchart (B)

Superior Product Performance

Complete Sealing

After aptamer modification and sealing, Hzymes warm-start reverse transcriptase has extremely low enzyme activity at non-working temperatures of 30°C/37°C/42°C, significantly outperforming competitor A in terms of sealing efficiency, which is more favorable for controlling non-specific amplification. At the working temperature (55°C), the activity can be fully released, having no impact on amplification performance. (Figure 2)

Figure 2. Hzymes warm-start reverse transcriptase sealing/activation effect diagram;

High Sensitivity

Warm-start characteristic, Hzymes warm-start reverse transcriptase can ensure the amplification of specific targets even at low template concentrations, with the amplification platform phase being consistent with high copy number templates (Figure 3A). Moreover, when comparing Hzymes’ complete warm-start reverse transcriptase product with competitor A in three different systems, Hzymes’ product performs better in terms of Ct value peak time and detection rate. (Figure 3B)

Figure 3. Comparison of Hzymes and competitor A warm-start reverse transcriptase performance; Figure A. Testing with high, medium, and low concentration templates in a certain all-in-one premixed system; Figure B. Testing of the complete product in different systems

Ultra-Fast

Hzymes warm-start reverse transcriptase has been modified to synthesize cDNA at a faster rate. Under consistent PCR programs, 2-minute reverse transcription can achieve the same sensitivity as 15-minute reverse transcription.

Figure 4. Amplification situation of Hzymes warm-start reverse transcriptase under different programs

Long Stability

In two RNA project all-in-one liquid premixed reaction liquids (RNA premixed, reaction liquid, enzyme liquid, primer probes premixed in advance in one tube), after 37°C pressure for 7 days, the performance of Hzymes warm-start reverse transcriptase is unaffected, while competitor A’s amplification performance is severely lagging, with sensitivity delayed by more than 2 Ct.

Figure 5. Hzymes warm-start reverse transcriptase accelerated performance

Application Cases

Hzymes warm-start reverse transcriptase has helped customers optimize and solve various problems, especially in the application of RNA all-in-one premixed stability, with specific case application effects shared as follows.

Compared to similar warm-start reverse transcriptases on the market, Hzymes warm-start reverse transcriptase can seal enzyme activity at higher temperatures, with higher sealing efficiency and stronger sealing stability, preventing the degradation of key components such as primer probes. It ensures high sensitivity and compatibility with fast programs, endowing all-in-one premixed reagents with higher stability.

Hzymes warm-start reverse transcriptase and competitor A were applied to RNA all-in-one premixed reagents (reaction liquid, enzyme liquid, multiple primer probes premixed in advance in one tube), treated at 37°C for 10 days, with newly prepared all-in-one premixed reagents as controls, tested under a 30-minute rapid program. The results show that the all-in-one premixed reagent prepared with Hzymes warm-start reverse transcriptase shows no significant difference from the newly prepared control group under pressure treatment conditions, while the all-in-one premixed reagent prepared with competitor A shows significantly reduced amplification efficiency (Ct values are later, fluorescence platform phase decreases) after pressure treatment.

Figure 6. Hzymes warm-start reverse transcriptase applied in all-in-one premixed reagents 37°C accelerated 10-day detection results (red curve is the accelerated curve)