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Recently , a research team from Xiamen University successfully developed a new fluorescent PCR technology, which improved the single-tube detection capability of fluorescent PCR by at least one order of magnitude, and promoted the development of mainstream fluorescent PCR instrument detection technology
.
The findings were published in the journal Proceedings of the National Academy of Sciences (PNAS)
.
Chemical machinery and equipment network hotspots focus on chemical machinery and equipment.
The findings were published in the journal Proceedings of the National Academy of Sciences (PNAS)
.
As the most mature and clinically widely used molecular diagnostic technology, PCR (polymerase chain reaction) technology has the advantages of high sensitivity, good specificity, timely and convenient, etc.
Screening, blood screening and other fields have great application value and have become the "gold standard" for many clinical diagnosis
.
During the epidemic, PCR technology has played an important role in the detection of viral nucleic acid, and its development has also received more attention
.
Screening, blood screening and other fields have great application value and have become the "gold standard" for many clinical diagnosis
.
During the epidemic, PCR technology has played an important role in the detection of viral nucleic acid, and its development has also received more attention
.
The development of PCR technology has been derived from multiplex PCR (multiplex PCR), real-time fluorescence quantitative PCR, digital PCR (digital PCR) and other technologies
.
Among them, the second generation technology - real-time quantitative PCR is the current mainstream
.
The technology monitors the fluorescent signal of amplified fragments in real time through specific fluorescent probes, and simultaneously realizes absolute gene expression and relative quantitative detection
.
At the same time, the closed-tube detection mode reduces the chance of contamination of amplification products, saves time and labor, and is conducive to the realization of automation
.
.
Among them, the second generation technology - real-time quantitative PCR is the current mainstream
.
The technology monitors the fluorescent signal of amplified fragments in real time through specific fluorescent probes, and simultaneously realizes absolute gene expression and relative quantitative detection
.
At the same time, the closed-tube detection mode reduces the chance of contamination of amplification products, saves time and labor, and is conducive to the realization of automation
.
However, the detection capability of real-time fluorescence quantitative PCR is limited by the number of instrument detection channels, and it is difficult to detect more than 6 target genes in a single reaction (2-4)
.
Adding a melting analysis step after PCR is an attempt by researchers to solve this problem.
Although the number of detectable target genes is increased to a certain extent, there are also problems such as increased detection cost and increased detection error rate
.
The limited number of target gene detection makes it difficult for real-time quantitative PCR technology to play a role in the detection of complex diseases involving multiple targets
.
.
Adding a melting analysis step after PCR is an attempt by researchers to solve this problem.
Although the number of detectable target genes is increased to a certain extent, there are also problems such as increased detection cost and increased detection error rate
.
The limited number of target gene detection makes it difficult for real-time quantitative PCR technology to play a role in the detection of complex diseases involving multiple targets
.
On February 24, 2022, a research team from the School of Life Sciences of Xiamen University published a research result in the journal Proceedings of the National Academy of Sciences (PNAS), reporting a single-step, multiple homogeneous phase called "MeltArray".
New technology of fluorescent PCR
.
New technology of fluorescent PCR
.
This technology utilizes the 5'-petal endonuclease activity of Taq DNA polymerase in the fluorescent PCR reaction, cleaves the "mediator probe" located downstream of the primer, releases the "mediator primer", and the "mediator primer" binds to the reaction system on the molecular beacon reporter probe in
.
Under the action of Taq enzyme polymerization activity, the "mediator primer" extends along the molecular beacon to generate a fluorescent double-strand with a specific melting point value
.
Since each molecular beacon can allow multiple "mediator primers" to form a series of fluorescent duplexes with different melting point values, the total number of target genes detectable by a single MeltArray multiplex PCR reaction is equal to the number of molecular beacons in the reaction multiplied by its The number of "medium primers" contained
.
.
Under the action of Taq enzyme polymerization activity, the "mediator primer" extends along the molecular beacon to generate a fluorescent double-strand with a specific melting point value
.
Since each molecular beacon can allow multiple "mediator primers" to form a series of fluorescent duplexes with different melting point values, the total number of target genes detectable by a single MeltArray multiplex PCR reaction is equal to the number of molecular beacons in the reaction multiplied by its The number of "medium primers" contained
.
To verify the multiplex detection capability of MeltArray, the researchers designed a variety of detection systems, including a 20-plex assay for human Y chromosome microdeletions, a 62-plex assay for determining E.
coli serotypes, a 24-plex assay for simultaneous identification and quantification of respiratory pathogens, a rectal KRAS mutation microsequencing analysis of cancer tissue samples corresponds to the three major application fields of molecular diagnosis of genetic diseases, infectious diseases and tumor molecular diagnosis, and has good sensitivity and specificity
.
coli serotypes, a 24-plex assay for simultaneous identification and quantification of respiratory pathogens, a rectal KRAS mutation microsequencing analysis of cancer tissue samples corresponds to the three major application fields of molecular diagnosis of genetic diseases, infectious diseases and tumor molecular diagnosis, and has good sensitivity and specificity
.
After the emergence of high-throughput sequencing technology, real-time quantitative PCR with a limited number of target gene detections is generally considered to be a "low-end" detection technology
.
MeltArray greatly increases the maximum number of target genes that can be detected by a single closed-tube real-time fluorescent PCR one-step method without changing the existing fluorescent PCR instruments, and promotes the "old technology" of fluorescent PCR to a "new level", filling the long-term There is a technical gap between low-level PCR and high-throughput detection
.
.
MeltArray greatly increases the maximum number of target genes that can be detected by a single closed-tube real-time fluorescent PCR one-step method without changing the existing fluorescent PCR instruments, and promotes the "old technology" of fluorescent PCR to a "new level", filling the long-term There is a technical gap between low-level PCR and high-throughput detection
.
(Source: Biological Center of the Ministry of Science and Technology, IVD Practitioner Network)
