Still struggling with doubts about constant temperature amplification technology? Don't be left behind!
Nucleic acid detection is the gold standard for pathogen detection, and qPCR is the most commonly used method for nucleic acid detection. However, large-scale screening puts forward new requirements for detection time. Compared with PCR, constant temperature amplification technology does not require temperature changes, and has obvious advantages in terms of amplification efficiency and equipment requirements.
In the face of mature traditional technologies, many researchers are worried about doubting the detection limit sensitivity of constant temperature amplification technology, and are in a wait-and-see state. However, at present, many companies have used the multi-enzyme constant temperature rapid nucleic acid amplification technology MIRA of AMP Future Biotech, and have obtained registration certificates for Class III medical devices, and the market for constant temperature amplification is expanding day by day.
The market application prospect of CRISPR detection technology is good, because it can not only detect various pathogens, but also play a great role in the detection of gene mutation and site-specific mutation. Its advantages are outstanding, and it is easier to combine with constant temperature detection technologies such as RPA and MIRA. It can not only improve the specificity but also give full play to the unique advantages of constant temperature technology. The detection time is short, the response is fast, and the sensitivity is high. The required detection equipment is low and the detection cost can be reduced.
The research group of Director Song Hongbin of the Center for Disease Control and Prevention of the Chinese People's Liberation Army published the article "Highly sensitive and rapid detection of SARS-CoV-2 via a portable CRISPR-Cas13a-based lateral flow assay" in the journal "JOURNAL OF MEDICAL VIROLOGY", with an impact factor of 20.693.
The key technologies used in the article are MIRA and CRISPR, which have been cleverly designed to achieve dual-target test strip detection
The constant temperature amplification technology MIRA combined with CRISPR immunochromatography double detection was used in the study. The detection of human internal reference genes is controlled by internal reference to ensure the effectiveness of sample collection, storage, transfer, and nucleic acid extraction.
Detection limit and specificity evaluation of MIRA combined with CRISPR immunochromatography dual detection method:
Detection limit: 0.25copies/uL, no cross-reaction with other 8 human coronaviruses with good detection specificity.
Detection limit and specificity evaluation of MIRA combined with CRISPR fluorescence detection method:
Detection limit: 0.25copies/uL, no cross-reaction with other 8 human coronaviruses with good detection specificity.
Due to the high sensitivity of this method, it may facilitate early screening of infected individuals. This study provides a powerful visualization tool for MIRA combined with CRISPR detection, which can significantly improve the reliability of on-site detection of SARS-CoV-2, and has great application value as a clinical detection.
High sensitivity: The CRISPR-LFA method established based on MIRA amplification technology showed a LoD of 0.25 copies/μl, and the detection limit broke through the LoD limit of 1 copies/μl of the pathogenic nucleic acid detection kits sold in my country, which is comparable to the RT-PCR method.
Fast and efficient: CRISPR immunochromatography can report detection results within 1 hour, so compared with RT-PCR, it can save 30 minutes to 1 hour;
Low detection cost: CRISPR immunochromatographic methods require minimal instrumentation and can provide a constant temperature of 37-42°C, saving costs associated with expensive instrumentation.
In the detection of viruses, fungi, bacteria, microorganisms, etc., the target nucleic acid is enriched by isothermal amplification technology, the amplified product binds to the Cas protein, activates the Cas protein to cut the reporter probe, and amplifies the detection signal. Due to this feature, many scientific research teachers are very interested in this research method.
AMP Future Bio and Sea Microbiology will launch an online live broadcast at 20:00 on Tuesday, July 25th - "Technical Application and Sharing of Constant Temperature Amplification and CRISPR Combination". The live broadcast will focus on introducing the multi-enzyme constant temperature nucleic acid amplification technology MIRA and CRISPR-Cas12a, cas13a, from the application scenarios, technical principles, and how to combine the two technologies with primer design, system preparation, and scientific research article cases (HPV multi-target typing detection, colloidal gold test strip type on-site rapid detection application, etc.), to provide detailed introductions for scientific research teachers.
