Development of Isothermal Amplification Technologies

Since the invention of PCR technology in 1980, nucleic acid detection based on PCR technology has been rapidly applied to clinical, food and environmental detection, and has become the gold standard for nucleic acid detection. However, PCR detection technology has many shortcomings, such as the large size of equipment, poor tolerance of crude samples, long reaction time, and difficulty in reaching single copy sensitivity. It has been difficult to meet the requirements of modern nucleic acid detection: fast speed (<30min), super sensitivity (1-5 copy/test), direct detection of a large volume of crude samples (20-50% sample volume of reaction), field operation, simplified operation, etc.

In the past 20 years, scientists have been trying to achieve the above goals through isothermal amplification, including Rolling Circle Amplification (RCA), Loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA), Nucleic Acid Sequence-Based Amplification (NASBA), Strand Displacement Amplification (SDA), Helicase-dependent Amplification (HDA), Transcription-mediated Amplification (TMA) and other isothermal amplification technologies. Among these isothermal amplification technologies, the LAMP method, which was first reported in 2000, is particularly attracting attention, and currently accounts for more than 60% of the isothermal amplification detection market. The reason is that other isothermal amplification technologies cannot achieve the comprehensive advantages of LAMP technology:

• The reaction speed is extremely fast. The optimized primer combination can detect a single copy molecule within 15 minutes, and the detection speed is close to or higher than RPA and NASBA.
• It can reach the limit of hypersensitivity detection. Molecules of 1-5 copies can be detected stably. It is difficult for any other isothermal amplification technology to reach such a stable level.
• The diversity of interpretation forms is suitable for rapid nucleic acid detection under various environments and conditions. LAMP amplification, as a terminal interpretation form, can be used to observe the detection results with the naked eye without any other auxiliary equipment. For HNB and OG orange green discoloration, the results can be observed with the naked eye. LAMP can also conduct real-time turbidity analysis with the aid of a turbidity meter. After SYBR Green fluorescent dye is added to the reaction system, a small constant temperature fluorometer can be used for real-time detection. These results can be interpreted in the field and other non-standard laboratories, and the required equipment is simple, compact, cheap, and easy to popularize. In addition, the fluorescent quantitative PCR instrument in the traditional laboratory can also be used for LAMP detection. SYBR Green, MB probe method, or LAMP TaqMan probe method can be used. For personnel who often operate PCR detection, it can be seamlessly connected without changing equipment.
• The undetected rate of the RNA virus is low. Because of the high mutation rate of the RNA virus, individual mutations in PCR detection have a greater impact on PCR amplification, which is easy to cause false negatives of the virus undetected. However, LAMP recognizes 8 segments of the target gene, and individual mutations in these recognition segments have little impact on LAMP amplification, so it is not easy to miss detection.
• The reagent is stable and easy to use. Compared with RPA, NASBA, and other technologies, LAMP and PCR adopt a single enzyme (or double enzyme) system for reaction, with a stable production batch, high-temperature resistance of the reaction enzyme system, and good reagent stability. The detection reagent only contains one tube of enzyme mix and one tube of primer/probe, which is convenient to use. It can easily conduct high-throughput detection on the fluorescent quantitative PCR machine. However, RPA, NASBA, and other systems require multiple complex enzymes, with strict proportion, difficult reagent stable production, harsh storage and transportation conditions, and difficult for high-throughput application.
• The LAMP method has the strongest tolerance to impurities. PCR, RPA, NASBA, and other amplification systems all need nucleic acid purification and preparation. In the direct amplification system for crude products, the sample loading is small (<10%) and cannot be loaded in large volumes. Impurities have a great impact on these amplification methods. The tolerance of the LAMP method to most samples is more than 20%, and the content of individual samples can be more than 50%. Such a large sample loading volume determines that LAMP has a higher detection rate.
• The amplification reaction is synchronized with virus inactivation. Since the LAMP reaction temperature is at 65°C, the virus can be inactivated during the direct expansion of the virus solution, reducing the contamination risk of consumables.
• High specificity. With the continuous improvement of the LAMP enzyme, reaction buffer, probe technology, and primer design concept, the non-specific amplification of LAMP has been improved qualitatively. At present, in the optimized LAMP system, the false positive situation has been fundamentally improved, and the proportion of false positives is close to or even lower than that of the PCR method.
• Application of multiple fluorescent probes. With the continuous improvement of the Bst polymerase and reaction buffer system and the invention of LAMP TaqMan technology, multiplex LAMP systems have been established. This multiplex LAMP technology has reached the same performance as the multi-target gene and internal reference quality control samples of the gold standard TaqMan PCR and meets the standard for clinical application.

Isothermal Amplification Platform of SBS Genetech

Based on the R&D experience of molecular tool enzymes and protein electronic reframing technology, SBS Genetech has established a comprehensive isothermal amplification core enzyme platform, including Bst DNA Polymerase, Bst Polymerase, and Bst DNA/RNA Polymerase.

Bst DNA/RNA Polymerase is at the core of our world-class platform

By carefully optimized reaction system, we have created PrimeIAmp™ series with excellent performance, high amplification efficiency and specificity. With freeze-drying technology, PrimeIAmp™ series can be transported and stored at room temperature. Diversified premixed chromogenic reagents meet different detection requirements, including OG color change (PrimeIAmp™ Basic Lyophilized Isothermal Amplification Microbeads), HNB color change (PrimeIAmp™ HNB Lyophilized Isothermal Amplification Microbeads), SYBR Green fluorescence method (PrimeIAmp™ SG Fluorescent Dye Lyophilized Isothermal Amplification Microbeads), etc.

Besides, we have also developed anti-contamination solution: PrimeIAmp™ Basic Isothermal Amplification MasterMix (with UDG). Thanks to the recognition of the dUTP substrate by Bst DNA/RNA Polymerase, the reaction substrate does not contain dTTP, so the amplified products are dUTP products, which can achieve anti-contamination in combination with heat-labile UDG. In the actual test, the amplification of contaminates was completely inhibited under the condition of containing 10⁵ copies of contaminates.

In addition, the isothermal amplification raw materials provided by SBS Genetech are not limited to LAMP amplification. They can also be used for isothermal amplification methods such as RCA (Rolling Circle Amplification), HDA (Helicase Dependent Amplification), SMAP (Smart Amplification Process), CPA (Crossing Priming Amplification), etc.