Molecular diagnostic technology is an indispensable part of modern medical testing, especially in pathogen detection, genetic disease screening and early diagnosis of cancer, where nucleic acid amplification technology plays a key role. PCR is currently the most widely used molecular diagnostic technique. However, due to its reliance on complex temperature control equipment and long reaction cycle, its application in some fields is limited. Therefore, isothermal amplification techniques (such as LAMP, NASBA, etc.) have gradually become alternative solutions, especially demonstrating unique advantages in on-site detection and rapid diagnosis.
In the past five years, the LAMP reaction has been the most highly regarded and fastest-growing technique in isothermal amplification. It has been used for the rapid diagnosis of influenza, tuberculosis, Zika virus, etc. However, the development of LAMP detection poses certain difficulties, especially in primer design and multiplex detection, which has brought considerable challenges to many researchers. So, is there a simpler and easier isothermal amplification to develop? In fact, it already existed before the LAMP technology emerged. This is the NASBA technology that this article focuses on introducing.
NASBA (Nucleic Acid Sequence-Based Amplification, nucleic acid sequence-based amplification) technology, as an isothermal nucleic acid amplification technology, does not rely on the temperature cycling required in traditional PCR technology. Instead, nucleic acid amplification is carried out through a series of enzymatic reactions at a constant temperature. The key advantage of this technology is that it can perform efficient nucleic acid amplification without relying on high temperatures, making it particularly suitable for on-site rapid detection.
The core principles of NASBA
NASBA technology is an isothermal nucleic acid amplification technique based on RNA templates. It first uses single-stranded RNA as a template and, under a constant temperature of 42 ° C, simulates the replication mechanism of reverse transcription disease in vivo through reverse transcriptase, RNase H, and T7 RNA polymerase, as well as forward and reverse primers to amplify the target RNA. The product can be obtained 10 to 12 times in 90 minutes, with the highest sensitivity reaching a single copy. Compared with immunological methods or other nucleic acid detection methods, NASBA technology has strong specificity and rapid detection. As the reaction product is single-stranded RNA, a hybridization detection system can be used to improve the sensitivity and specificity of this technology.
Reaction components:
Template RNA
Primer pair: Two specific primers
First primer (P1) : carries the T7 RNA polymerase promoter sequence at the 5 'end.
Second primer (P2) : Complementary to the downstream sequence of the target RNA.
Three enzymes
AMV reverse transcriptase: Used for cDNA synthesis
RNase H: Degrading RNA: RNA in DNA hybridization chains
T7 RNA polymerase: Transcribe RNA amplification products from double-stranded DNA templates
Nucleotides (dNTPs and NTPs
Buffer: Optimize enzyme activity
Reaction stepsThe NASBA reaction is divided into the following stages:
Initial denaturationThe RNA secondary structure was uncoiled at 63-65℃ for 20 minutes
2. Reverse transcription
Target RNA and primer binding: The template RNA binds to the first primer P1, which contains the T7 promoter sequence
Synthesis of the first-stranded cDNA: AMV uses RNA templates, starting from P1 primers, to synthesize single-stranded cDNA complementary to RNA
3. RNA degradation
RNase H function: RNase H specifically degrades RNA: RNA strands in DNA hybridization molecules, leaving single-stranded cDNA
4. Synthesis of the second-strand cDNA
Second primer P2 binding: The P2 primer binds to the complementary region of the single-stranded cDNA
Extension: AMV reverse transcriptase starts to synthesize the second DNA strand from the P2 primer, forming double-stranded DNA
5. RNA amplification
T7 RNA polymerase plays a role: T7 RNA polymerase recognizes the T7 promoter sequence in the double-stranded DNA template and transcribe it as a template to generate a large number of RNA molecules
Cyclic amplification: The newly generated RNA molecules can be used as templates to re-enter the above cycle and further amplify the products. The amplification cycle starts from 2-5 and is carried out at 41°C.
Reaction characteristics
Amplify RNA directly
IsothermalThe reaction takes place at a constant temperature (typically 41°C) without the need for thermal cycling equipment.
Although NASBA has an initial denaturation step, it is still regarded as an isothermal amplification reaction because it does not require cycling at high and low temperatures like PCR.
High specificityThe T7 RNA polymerase specifically recognizes the T7 promoter, and the double primer design ensures the specificity of amplification
Linear RNA productUnlike the exponential amplification generated by PCR, NASBA produces linearly amplified RNA products.
NASBA product testing
Although the amplification product of NASBA is RNA, which is different from the DNA amplification product of LAMP, the detection method of NASBA products is almost the same as that of LAMP, specifically including:
1.Fluorescence probe methodFluorescent probes (such as Taqman probes) bind to the target RNA sequence during amplification.The fluorescence signal will only be released when the probe binds to the target RNA sequence and is degraded by T7 RNA polymerase.Monitor the increase of products in real time.
2.Gel electrophoresis methodThe amplification products were separated by gel electrophoresis, and the difference in molecular size was utilized to determine whether the target sequence had been successfully amplified. Gel electrophoresis can be visualized using DNA/RNA dyes such as EB dye or SYBR Green.
3.thanColor methodDetection is carried out by using the color change resulting from the reaction with the amplification product. For instance, when using probes or dyes with enzyme tags, after the amplification reaction is completed, the substrate is added to produce a color reaction. The change in color represents the presence and quantity of RNA, which is applicable to POC levels
4.Fluorescent dye methodChimeric fluorescent dyes (such as SYBR Green) can bind to double-stranded RNA and emit fluorescence signals after excitation. Quantitative analysis of amplification products can be achieved by real-time detection of the intensity changes of fluorescence signals.
5.electricityChemical methodQuantitative analysis is achieved by detecting the influence of amplification products on electrochemical signals. Electrochemical sensors can sense changes in ions, current or potential during the reaction process, thereby inferring the concentration of the target RNA.
6.sideRadial chromatographyThe RNA products were captured by colloidal gold or fluorescence method and developed on the test strips
Different detection techniques each have their own advantages. The choice of an appropriate detection method depends on the requirements of the experiment. For instance, fluorescent probe detection is often used for highly sensitive real-time monitoring, gel electrophoresis is suitable for product validation in the basic research stage, while colorimetry and fluorescent dye staining are mostly employed for on-site rapid detection. By integrating these technologies, NASBA amplification can achieve precise and efficient molecular diagnosis and is widely applied in fields such as pathogen detection, environmental monitoring, and biomarker analysis.
The Development history of NASBA
NASBA in molecular biologyThe application history of this field is long and profound, even earlier than that of LAMP。
1Origin:
In 1991, it was proposed and successfully developed by A research team from Eindhoven University of Technology in the Netherlands (by Willem A. J. H. van der Velden et al.), and since then, RNA amplification detection no longer relies on traditional PCR.
2. Development History:From basic researchIt has been widely applied
In the first few years, NASBA is mainly used in laboratory research, especially in fields such as gene expression analysis and viral load monitoring, such as HIV and HBV. In 1992, scientists further optimized the NASBA technology to ensure its stable operation under various experimental conditions, especially when using a wide range of sample sources such as blood, urine, saliva, etc. NASBA has gradually become an important research tool and has become more efficient and adaptable through continuous optimization of its enzyme system and reaction conditions.
3. Technological progressCompared with other nucleic acid amplification techniquesThe combination
With the rapid development of molecular diagnostic techniques, NASBA technology is also constantly innovating and improving. Compared with traditional PCR, an important advantage of NASBA is that it can perform isothermal amplification without temperature control equipment and has lower energy consumption than LAMP reactions. This makes it suitable in resource-limited environments, especially inPOCTIts application in on-site detection has been rapidly promoted.
At the beginning of the 21st centuryNASBA has begun to be combined with microfluidic technologyForm a more innovative diagnostic platform. These integrated systems, by combining NASBA amplification technology with automated microfluidic chips and sensor technology, make detection more sensitive and convenient, and enable real-time monitoring of the amplification process. Compared with traditional PCR equipment, this miniaturized device not only reduces costs but also enhances the convenience and operability of detection.
4. Market ApplicationFrom research to clinical practiceThe bed
With the continuous maturation of technology, NASBA is beginning to enter a broader rangeThe market application. In the late 1990s and the early 21st centuryMany biotech companies have commercialized NASBA technologyAnd apply it toClinical testing, environmental monitoring, food safety and other fields. Especially in virus detection andIn terms of microbiological testing, NASBA has become an indispensable technical tool.
For instance, in the early detection of viral infections such as HIV, HCV, and influenza, NASBA can provide more sensitive test results than PCR and has a shorter reaction time. This has enabled it to be widely used in epidemic prevention and control, early diagnosis and treatment monitoring"General application.
In addition, NASBA technology is also applied toIt has become a multi-purpose nucleic acid amplification technology in fields such as food safety testing, water quality monitoring and environmental pollution monitoring.
5. Current and Future Development:Multi-field applications and further innovationnew
After entering the 2020s, NASBA technology has continued to innovate and improve. With the rapid development of molecular biology, nanotechnology, microfluidic technology and smart devices, NASBA is no longer confined to the traditional field of molecular diagnosis, but has also been combined with these emerging technologies, further expanding its application scenarios. For instance, by integrating nanosensor technology, NASBA can directly provide quantitative results in rapid detection, rather than merely qualitative detection.
Furthermore, with the rapid development of genomics and precision medicineNASBA has also been used to help achieve personalized medicine. By detecting specific gene mutations or pathogens, NASBA can provide precise pathological information, assisting doctors in customizing personalized treatment plans for patients.
NASBA technical advantages
1. High sensitivity and high specificity：NASBA can effectively detect target nucleic acids as low as the copy level, and thus has wide applications in pathogenic microorganism detection, virus detection, and other fields.
2. Isothermal operationCompared with PCR, NASBA does not require complex temperature control equipment and can complete the reaction at room temperature. Compared with LAMP detection, it has a lower reaction temperature and lower requirements for energy consumption and equipment, which gives it a unique advantage in convenience and makes it particularly suitable for on-site immediate detection.
3. Rapid amplificationBecause it does not rely on temperature cycling, the amplification reaction of NASBA is faster than that of traditional PCR technology, and nucleic acid testing can usually be completed within 30 minutes.
4. Strong adaptabilityNASBA can adapt to many thingsDifferent types of samples have stronger tolerance to inhibitors in the samples, and thus can play a role in a variety of clinical and environmental tests.
NASBA Application
NASBA technology in molecular diagnosisApplications in the process of interruption include
1. Rapid detection of pathogens:For instance, for nucleic acid testing of viruses such as HIV, influenza, HBV, HCV and the novel coronavirus, NASBA technology can provide accurate results in a short time, and is particularly suitable for rapid screening and on-site testing.
2. Environmental monitoringFor instance, in water source monitoring, NASBA can be used to detect pathogenic microorganisms in water bodies. By conducting rapid and sensitive detection of environmental samples, it effectively safeguards public health.
3. On-site Testing and POCTNASBA has lower requirements for equipment reaction temperatures than LAMP, making it more suitable for areas with limited resources or emergency situations. NASBA can provide doctors with immediate diagnostic results and help them make quick decisions.
4. Personalized Medicine and Precision MedicineWith the development of precision medicine, the role of NASBA in individualized medicine has gradually become prominent. By detecting specific genetic markers or pathogens, NASBA technology can help doctors provide tailor-made treatment plans for patients.
NASBA's Future outlook
With the continuous advancement and optimization of technology, NASBA is expected to play a role in more fields. Its potential in on-site detection, rapid diagnosis and personalized treatment will further promote the development of molecular diagnostic technology. In addition, by integrating other innovative technologies such as microfluidic chips and smartphone platforms, the application scenarios of NASBA will become more extensive, providing more possibilities for the early detection and treatment of diseases.
In China, PCR technology has developed very maturely, with a high degree of standardization and a complete supply chain and regulatory system. As the leading technology in the IVD market, it has to a considerable extent restricted the popularization of NASBA technology. In fact, NASBA technology has a long history internationally. However, in the Chinese market, many IVD enterprises and laboratories have limited understanding of NASBA technology, which leads to a restricted market awareness of it and the fact that the unique advantages of NASBA have not been fully exploited.
Although the promotion of NASBA technology in China's IVD market faces multiple challenges, with the increasing demand for rapid RNA amplification and the integration of NASBA technology with emerging technologies such as CRISPR, this technology may still have its unique development space in the future, especially in specific pathogen detection or resource-limited scenarios.
This article is reprinted. Source: Maidian Biotechnology
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