Introduction to the principle, characteristics and application fields of real-time fluorescent quantitative PCR
Polymerase Chain Reaction (PCR) can perform exponential amplification of specific nucleotide fragments. After the end of the amplification reaction, we can qualitatively analyze the amplified product by gel electrophoresis, or quantitatively analyze it by radionuclide incorporation after labeling. Whether qualitative or quantitative, the end products of the PCR are analyzed. But in many cases, we are interested in the amount of starting template before amplification by PCR signal. For example, we want to know the copy number of a transgenic animal or plant transgene or the expression of a particular gene in a particular tissue. Under this demand, real-time PCR technology came into being.
Real-time quantitative polymerase chain reaction (Real Time PCR) is a nucleic acid quantification technology developed on the basis of qualitative PCR technology. Real-time PCR technology was introduced in 1996 by Applied Biosystems, Inc., and a fluorescent group was added to the PCR reaction system to monitor the entire PCR process in real time using fluorescence signal accumulation, making each cycle "visible" and finally passing Ct value and Standard curve A method for quantifying the initial concentration of DNA (or cDNA) in a sample. Real-time PCR is currently the most sensitive and accurate method for determining DNA (or cDNA) copy number in a sample. If used for RNA detection, this is called reverse transcription real-time PCR (Real-time RT-PCR) is a real-time PCR method, which refers to DNA or reverse-input (RT-PCR) RNA through the polymerase chain The amplification process of the DNA is monitored and monitored in real time, and the amplification product is measured during the exponential growth phase of the amplification, since the amplification index growth period measurement correlates with the specific amount of specific DNA (RNA) to achieve quantitative detection. The basic goal of RealTime PCR is to accurately measure and identify very small amounts of specific nucleic acids, so that the amount of the original target gene can be quantified by monitoring the CT value. The biggest advantage of real-time PCR is to overcome the large error of quantification of the end-point PCR method after entering the plateau or saturation period, and to achieve accurate quantification of DNA/RNA. This technology not only realizes the quantification of DNA/RNA template, but also has the characteristics of high sensitivity and specificity, multiple reactions, high degree of automation, no pollution, real-time and accurate. The technology is used in medical clinical testing and clinical medical research. It has important meaning.
Technical characteristics of microRNA real-time fluorescent quantitative PCR
Although microRNA real-time quantitative PCR is a recently developed detection technology, its technical details are still being improved, but the microRNA real-time quantitative PCR detection system has the following advantages over other microRNA detection technologies:
Highly specific, it can quantify only mature microRNAs, effectively distinguishing mature microRNA molecules and precursor molecules as well as homologous molecules of other mature microRNAs;
Fast, simple, and time-saving, the entire operation takes only two steps, and in less than three hours, you can get high-quality data;
High detection sensitivity, low sample consumption, only 1-10ng of total RNA or about 50pg of microRNA;
The ultra-wide linear range spans seven orders of magnitude.
System composition and working principle of real-time quantitative PCR
The fluorescence quantitative detection system consists of real-time fluorescence quantitative PCR instrument, real-time fluorescence quantitative reagent, general-purpose computer, and automatic analysis software. The device consists of a fluorescence quantification system and a computer to monitor the fluorescence of the cycle.
A computer connected to a real-time device collects fluorescence data. The data is displayed graphically through the developed real-time analysis software. Raw data is plotted as a plot of fluorescence intensity versus number of cycles. The analysis can be started after the raw data is collected. The software of the real-time device enables the collected data to be normalized to compensate for differences in background fluorescence. The domain value level can be set after normalization, which is the level at which the fluorescence data is analyzed. The number of cycles that a sample experiences when it reaches the threshold value level is called the Ct value (the number of cycles of the limit point). The domain value should be set to maximize the amplification efficiency of the exponential phase so that the most accurate and repeatable data can be obtained. If the standard is also amplified at the same time, linear regression analysis will generate a standard curve that can be used to calculate the concentration of the unknown sample.
The so-called Real-time Q-PCR technology refers to a method in which a fluorescent gene is added to a PCR reaction system, the whole PCR process is monitored in real time by using fluorescence signal accumulation, and the unknown template is quantitatively analyzed by a standard curve. In the development of real-time technology, two important discoveries play a key role:
- In the early 1990s, the discovery of the 5' exonuclease activity of Taq DNA polymerase, which degraded specific fluorescent probes, made it possible to detect PCR products indirectly.
- The use of a fluorescent double-labeled probe thereafter allows the entire reaction to be monitored in real time in a closed reaction tube. The combination of these two findings and the commercialization of the corresponding instruments and reagents have led to the use of real-time Q-PCR methods in research work.
Gradient function of quantitative PCR instrument: For quantitative PCR reaction using dye method, although there are various PCR primer design software or empirical formula to calculate melting temperature (Tm value), the formula used is different, the primer sequence is different, and the Tm value is used. The difference will be great. The melting temperature of the primer determines the annealing temperature. Moreover, the combination of bases in the template is ever-changing. For special fragments, the data obtained by the empirical formula may not be able to produce accurate results. The slight difference in annealing temperature may have a decisive influence on the results, so the "conditions" are once Very headache. The appearance of gradient PCR solves some problems. During the reaction, the temperature control conditions of each well can be changed according to the gradient within the specified range. According to the results, the most suitable reaction conditions can be found in one step.
Not only the annealing temperature, but also the denaturation temperature and extension temperature can be optimized - this is very important for the amplification of many polymerase mixed enzymes such as Invitrogen, Clontech, Promega, most of the high-fidelity Taq enzymes, because Taq and correcting enzymes are optimal. The reaction temperature may vary significantly, and optimizing the extension temperature is important. Fluorescence quantitative PCR with gradient function can complete the optimization process that can be completed in many experiments in the past, which simplifies the cumbersome experiment of exploring the PCR reaction conditions, saves the experiment time and improves the efficiency, and saves the experiment cost.
The copy number of DNA generated during the PCR reaction increases exponentially. As the number of reaction cycles increases, the final PCR reaction no longer generates an exponential template and enters the plateau. In conventional PCR, gel electrophoresis is used to separate and fluorescent staining is used to detect the final amplification product of the PCR reaction, so the quantification of the PCR product by this endpoint method is unreliable. In real-timeQ-PCR, the entire PCR reaction amplification process is monitored in real time and the fluorescence signal associated with the amplification is continuously analyzed. As the reaction time progresses, the detected fluorescence signal changes can be plotted as a curve. . In the early stages of the PCR reaction, the level of fluorescence produced cannot be clearly distinguished from the background, and the generation of post-fluorescence enters the exponential phase, the linear phase, and the final plateau, so that the amount of PCR product can be detected at some point in the exponential phase of the PCR reaction. And thereby infer the initial content of the template.
In order to facilitate comparison of the detected samples, in the exponential phase of the real-time Q-PCR reaction, the domain value of a certain fluorescent signal is first set. Generally, this threshold is the fluorescent signal of the first 15 cycles of the PCR reaction. As a fluorescent background, the default setting of the fluorescence domain value is 10 times the standard deviation of the fluorescent signal of 3 to 15 cycles. If a fluorescent signal is detected that exceeds the domain value is considered a true signal, it can be used to define the number of domain value cycles (Ct) for the sample. The meaning of the Ct value is the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set domain value. Studies have shown that there is a linear relationship between the Ct value of each template and the logarithm of the initial copy number of the template. The more the starting copy number, the smaller the Ct value. A standard curve can be made using a standard of known starting copy number, so the initial copy number of the sample can be calculated from the standard curve as long as the Ct value of the unknown sample is obtained.
1, the definition of Ct value
In the real-time PCR technique, there is a very important concept - Ct value. C stands for Cycle, and t stands for threshold. The meaning of Ct value is the number of cycles experienced by the fluorescent signal in each reaction tube when it reaches the set domain value.
2, the setting of the fluorescence field value (threshold)
The fluorescence signal of the first 15 cycles of the PCR reaction is used as the fluorescence background signal. The default setting of the fluorescence domain value is 10 times the standard deviation of the fluorescence signal of 3-15 cycles, ie: threshold=10'SDcycle6-15
3. Relationship between Ct value and starting template
Studies have shown that there is a linear relationship between the Ct value of each template and the logarithm of the starting copy number of the template [1]. The more the starting copy number, the smaller the Ct value. A standard curve can be made using a standard of known starting copy number, where the abscissa represents the logarithm of the starting copy number and the ordinate represents the Ct value. Therefore, as long as the Ct value of the unknown sample is obtained, the initial copy number of the sample can be calculated from the standard curve.
4. Fluorescence chemistry
The fluorescence chemistry used in real-time PCR can be divided into two types: fluorescent probes and fluorescent dyes. The principle is now briefly described as follows:
1) TaqMan fluorescent probe : When PCR is added, a specific fluorescent probe is added at the same time as adding a pair of primers. The probe is an oligonucleotide, and a reporter fluorophore and a quenching are respectively labeled at both ends. Fluorescent group. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; when PCR is amplified, the 5-3 exonuclease activity of the Taq enzyme degrades the probe, allowing the reporter to fluoresce and quench the fluorescence. The groups are separated so that the fluorescence monitoring system can receive the fluorescent signal, that is, one fluorescent molecule is formed for each DNA strand amplified, and the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product. The new TaqMan-MGB probe enables this technology to perform both quantitative and synthetic gene mutations (SNPs), and is expected to be the technology platform of choice for gene diagnosis and individualized drug analysis.
2) SYBR fluorescent dye : In the PCR reaction system, an excessive amount of SYBR fluorescent dye is added. After the SYBR fluorescent dye is specifically incorporated into the DNA double strand, the fluorescent signal is emitted, and the SYBR dye molecule not incorporated into the chain does not emit any fluorescence. The signal ensures that the increase in fluorescence signal is fully synchronized with the increase in PCR product.
Application fields of real-time fluorescent quantitative PCR technology
Real-time PCR is a leap forward in DNA quantification. Using this technology, we can perform quantitative and qualitative analysis of DNA and RNA samples. Quantitative analysis includes both absolute quantitative analysis and relative quantitative analysis. The former can obtain the copy number and concentration of the gene in a sample; the latter can compare the gene expression levels in the two samples processed in different ways. In addition, we can qualitatively analyze PCR products or samples: for example, using fusion curve analysis to identify amplification products and primer dimers to distinguish non-specific amplification; using specific probes for genotyping and SNP detection Wait. At present, real-time fluorescent PCR technology has been widely used in basic scientific research, clinical diagnosis, disease research and drug research and development. The most important applications focus on the following aspects:
- Absolute quantitative analysis of DNA or RNA. Including detection of pathogenic microorganisms or virus content, detection of transgenic plant and animal copy number, detection of RNAi gene inactivation rate, etc.
- Differential analysis of gene expression. For example, comparing differences in expression of specific genes between different processed samples (such as drug treatment, physical treatment, chemical treatment, etc.), differences in expression of specific genes at different phases, and confirmation of cDNA or differential results.
- Genotyping. For example, SNP detection, methylation detection, and the like.
The application of real-time PCR technology in medical applications:
Pathogen detection :
Currently, quantitative PCR detection technology can be used to quantify pathogens such as Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Human papillomavirus, Herpes simplex virus, Hepatitis virus, Mycobacterium tuberculosis, Parvovirus B19, Epstein-Barr virus and human cytomegalovirus. Determination. Compared with the traditional detection method, it has the advantages of high sensitivity, less sampling, quick and easy.
Genetic and prenatal and postnatal diagnosis :
So far, people have been unable to treat hereditary diseases caused by hereditary substance changes. They can only reduce the birth of sick animals through prenatal monitoring to prevent the occurrence of various genetic diseases, such as reducing X-linked genetic diseases. Birth, separation of fetal DNA from pregnant women's peripheral blood, real-time fluorescent quantitative PCR detection of its Y sex determination region gene is a non-invasive method, easy for pregnant women to accept. Exceptions can also be detected by real-time fluorescent quantitative PCR for Toxoplasma gondii and syphilis in pregnant women, which can provide a powerful help for finding the cause of unexplained abortion and habitual abortion, and greatly improve the prenatal and postnatal care.
Guide treatment :
Quantitative analysis of pathogens showed that the amount of pathogens was related to the efficacy of certain drugs. Such as high level of HCV expression, interferon treatment is not sensitive, while HCV low titer, interferon sensitive; in the lamivudine treatment, HBV-DNA serum content has decreased, and then if it rises again or beyond Level, it indicates that the virus has changed.
Tumor gene detection :
Although the mechanism of tumor onset is not yet clear, mutations in related genes are widely accepted as the underlying cause of carcinogenic transformation. Increased expression and mutation of the p53 oncogene can occur in many early stages of the tumor. Real-time PCR is not only effective in detecting gene mutations, but also accurately detecting the expression of oncogenes. With the continuous discovery of new genes associated with tumors, real-time PCR technology will play a greater role in tumor research.
Immune detection :
B27, HLA, etc. were detected by real-time PCR to help diagnose immune diseases such as ankylosing spondylitis, rheumatoid arthritis, and organ transplant rejection.
Application of real-time PCR technology in drug research:
New drug development research,
Human drugs and other drugs : drugs for some infectious diseases, such as various viral diseases, bacterial diseases, etc. In the development of new drugs, quickly understanding the impact of drugs on the disease process can save a lot of manpower and time for the development of new drugs. And the funds, compared with the previous Elisa and other methods, real-time fluorescent quantitative PCR technology can quickly, accurately, quantitatively and sensitively determine the content of pathogens in blood or tissue, so the effect of using analytical drugs, to compare different formulations The efficacy, dosage, time of administration, etc. provide rapid and quantitative evaluation.
Research and development of drugs and therapies for ultra-early infections :
Real-time quantitative PCR method is the determination of the amount of viral nucleic acid in the blood, so it is not necessary to wait for the production of antibodies in patients. At the same time, because its sensitivity is inconsistent with that of Elisa, in the early stage of viral infection, that is, when the virus content in the blood is very low. It can be measured. Therefore, a new field has emerged, namely, how to use the medicine when the virus or bacteria content is very low, what kind of medicine and the amount of medicine to be used for research, and contribute to the eradication of the disease in the early stage.
Drug efficacy study :
For some new drugs that have already been marketed or old drugs that have been used for a long time, the effects of the drugs, the amount of drugs used and the time of administration can be further studied, and further research is carried out for the rational use of these drugs for the benefit of mankind. The methods used in the past are not accurate enough, and the sensitivity is not enough. Therefore, there is a lot of content that needs to be studied. This aspect requires a lot of research content and great significance.
Research on new post-reduction indicators :
For infectious diseases, after the drug has reached a certain level, it is considered that the drug can be discontinued, but what should be discontinued. Currently, there is no clear indicator. The indicators used now are limited by the sensitivity and accuracy of the test method. This indicator may not be reasonable, so it is necessary to conduct further research on the indicators of cure and the relationship between the index and the recurrence rate and the conversion of the disease into other diseases, so as to lay a solid theoretical foundation for the complete cure of the disease by drugs or therapies.
Development of new diagnostic and test reagents :
Many existing diagnostic and testing methods can not meet the requirements of rapid, sensitive and quantitative, such as the existing culture method, Elisa method, etc., and the quantitative PCR method can do this, which can be used for clinical diseases and commodity inspection. , grain and oil inspection, food inspection, blood testing, etc. to develop new reagents, thereby improving the sensitivity, speed and accuracy of the inspection; the types of such reagents are very many, the social and economic benefits of the developed reagents are Very huge.
Application of Real-time Fluorescence Quantitative PCR in Detection of Human Infected A(H1N1) Swine Influenza Virus:
In April 2009, the global public health incident of swine flu was developing rapidly, and there were many countries. The World Health Organization raised the global influenza pandemic warning level from 4 to 5 on the evening of the 29th. China also on the 28th. The State Council executive meeting was held to deploy prevention and control. It has now entered the key implementation phase of prevention and control measures, where detection and diagnostic techniques are essential.
After the human swine flu epidemic, the US Centers for Disease Control and Prevention (CDC) issued a temporary guideline for the treatment and prevention of human swine influenza virus on April 28, 2009, which provides a diagnostic method for confirmed cases: an acute fever respiratory tract. The clinical symptoms of the disease were confirmed by real-time real-time PCR (PCR) or viral culture laboratory test methods to confirm infection with the A(H1N1) swine influenza virus.
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