Quantitative Real-time PCR is a method of measuring the total amount of product after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction using fluorescent chemicals. A method for quantitative analysis of specific DNA sequences in a sample to be tested by an internal or external reference method.
Real-time PCR is the real-time detection of the PCR process through the fluorescent signal during the PCR amplification process. Since there is a linear relationship between the Ct value of the template and the initial copy number of the template in the exponential period of PCR amplification, it becomes the basis for quantification.
Principle of PCR technology
The so-called real-time quantitative PCR technology refers to the method of adding fluorescent groups to the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and finally quantitatively analyzing the unknown template through the standard curve.
1. SYBR Green I method:
In the PCR reaction system, excess SYBR fluorescent dye is added. After the SYBR fluorescent dye is specifically incorporated into the DNA double-strand, it emits a fluorescent signal, while the SYBR dye molecule that is not incorporated into the chain will not emit any fluorescent signal, thus ensuring the fluorescent signal The increase was completely synchronized with the increase in PCR product.
2.TaqMan probe method:
When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the 5′-3′ exonuclease activity of Taq enzyme cleaves and degrades the probe, making the reporter fluorophore and the quencher group. The fluorescent group is separated, so that the fluorescence monitoring system can receive the fluorescent signal, that is, each time a DNA chain is amplified, a fluorescent molecule is formed, and the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product.
After the Taqman probe labeled with fluorescein is mixed with the template DNA, the thermal cycle of high temperature denaturation, low temperature renaturation, and suitable temperature extension is completed, and the Taqman probe that is complementary to the template DNA is cut off according to the law of polymerase chain reaction. Fluorescein is free in the reaction system and emits fluorescence under specific light excitation. With the increase of the number of cycles, the amplified target gene fragments increase exponentially. By real-time detection of the corresponding fluorescence signal intensity that changes with the amplification , to obtain the Ct value, and at the same time, using several standards with known template concentrations as a control, the copy number of the target gene in the sample to be tested can be obtained.
The meaning of Ct value (Cycle threshold, cycle threshold) is: the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold
1. Fluorescence threshold (threshold) setting
The fluorescence signal of the first 15 cycles of the PCR reaction is used as the fluorescence background signal, and the default (default) setting of the fluorescence threshold is 10 times the standard deviation of the fluorescence signal of the 3-15 cycles, namely: threshold = 10*SDcycle 3- 15
2. Relationship between Ct value and starting template
The Ct value of each template has a linear relationship with the logarithm of the initial copy number of the template, and the formula is as follows.
n is the number of cycles of the amplification reaction, X0 is the initial template amount, Ex is the amplification efficiency, and N is the amount of amplified product when the fluorescent amplification signal reaches the threshold intensity.
The higher the initial copy number, the lower the Ct value. A standard curve can be made using a standard with a known initial copy number, where the abscissa represents the logarithm of the initial copy number, and the ordinate represents the Ct value. Therefore, as long as the Ct value of an unknown sample is obtained, the starting copy number of the sample can be calculated from the standard curve.
The fluorescent substances used in real-time quantitative PCR can be divided into two types: fluorescent probes and fluorescent dyes. The principle is briefly described as follows:
1. TaqMan fluorescent probe: a specific fluorescent probe is added along with a pair of primers during PCR amplification. The probe is an oligonucleotide, and both ends are labeled with a reporter fluorescent group and a quencher. Fluorophore. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the 5′-3′ exonuclease activity of Taq enzyme cleaves and degrades the probe, making the reporter fluorophore and the quencher group. The fluorescent group is separated, so that the fluorescence monitoring system can receive the fluorescent signal, that is, a fluorescent molecule is formed every time a DNA chain is 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 gene analysis and gene mutation (SNP) analysis, and is expected to become the preferred technology platform for genetic diagnosis and personalized medicine analysis.
2. SYBR fluorescent dye: In the PCR reaction system, adding excess SYBR fluorescent dye, SYBR fluorescent dye is non-specifically incorporated into the DNA double-strand, and emits a fluorescent signal, while the SYBR dye molecule that is not incorporated into the strand will not emit any fluorescence. signal, thereby ensuring that the increase in fluorescent signal is completely synchronized with the increase in PCR product. SYBR binds only to double-stranded DNA, so a melting curve can be used to determine whether a PCR reaction is specific.
3. Molecular beacon: It is a stem-loop double-labeled oligonucleotide probe that forms a hairpin structure of about 8 bases at the 5 and 3 ends. The nucleic acid sequences at both ends are complementary paired, resulting in the fluorophore and The quencher groups are in close proximity and do not fluoresce. After the PCR product is generated, during the annealing process, the middle part of the molecular beacon is paired with a specific DNA sequence, and the fluorescent gene and the quenching gene are separated to produce fluorescence  .
Traditional quantitative PCR
1. Introduction to Traditional Quantitative PCR Methods
1) Internal reference method: add the quantified internal standard and primers to different PCR reaction tubes, and the internal standard is synthesized by genetic engineering method. The upstream primers are fluorescently labeled, and the downstream primers are not. At the same time as the template is amplified, the internal standard is also amplified. In the PCR product, due to the different lengths of the internal standard and the target template, the amplification products of the two can be separated by electrophoresis or high performance liquid phase, and their fluorescence intensities can be measured respectively, and the internal standard can be used as a control to quantify the template to be detected.
2) Competition method: select an exogenous competitive template containing a new endonuclease site produced by mutant clones. In the same reaction tube, the sample to be tested and the competing template are simultaneously amplified with the same pair of primers (one of the primers is fluorescently labeled). After amplification, the PCR product is digested with endonuclease, and the product of the competitive template is digested into two fragments, while the template to be tested is not digested by enzyme. The two products can be separated by electrophoresis or high performance liquid phase, and the fluorescence intensity can be measured separately , based on the known template to infer the initial copy number of the unknown template.
3) PCR-ELISA method: using labeled primers such as digoxigenin or biotin, the amplified product is bound by a specific probe on the solid phase plate, and then anti-digoxigenin or biotin enzyme-labeled antibody-horseradish peroxidation is added. Substrate enzyme conjugate, and finally the enzyme develops the color of the substrate. The conventional PCR-ELISA method is only a qualitative experiment. If an internal standard is added to make a standard curve, the purpose of quantitative detection can also be achieved.
2. The role of internal standards in traditional quantification
Because the traditional quantitative methods are end-point detection, that is, the detection is performed after PCR reaches the plateau phase, and when PCR reaches the plateau phase through logarithmic phase amplification, the detection reproducibility is extremely poor. The same template was repeated 96 times on a 96-well PCR machine, and the results obtained were very different, so it was impossible to directly calculate the amount of the starting template from the amount of the end product. Inaccuracies caused by the quantification of the final product can be partially eliminated by adding an internal standard. But even so, traditional quantitative methods can only be regarded as semi-quantitative and rough quantitative methods.
3. Influence of Internal Standard on Quantitative PCR
If an internal standard with a known initial copy number is added to the sample to be tested, the PCR reaction becomes double PCR, and there is interference and competition between the two templates in the double PCR reaction, especially when the initial copy number of the two templates When the difference is relatively large, this competition will be more significant. However, since the initial copy number of the sample to be tested is unknown, an appropriate amount of known template cannot be added as an internal standard. It is also for this reason that although the traditional quantitative method adds an internal standard, it is still only a semi-quantitative method.
Real-time fluorescence quantitative PCR technology effectively solves the limitation of traditional quantitative only end-point detection, realizes the detection of the intensity of the fluorescent signal once in each cycle, and records it in the computer software, through the calculation of the Ct value of each sample, Quantitative results were obtained according to a standard curve. Therefore, real-time quantitative PCR without internal standard is based on two foundations:
1) Reproducibility of Ct value The PCR cycle has just entered the true exponential amplification phase (logarithmic phase) when it reaches the cycle number where the Ct value is located. At this time, the slight error has not been amplified, so the reproducibility of the Ct value is excellent. , that is, the same template is amplified at different times or amplified in different tubes at the same time, and the obtained Ct value is constant.
2) Linear relationship between Ct value and starting template Since there is a linear relationship between the Ct value and the logarithm of the starting template, the standard curve can be used to quantitatively determine the unknown sample. Therefore, real-time fluorescence quantitative PCR is a quantitative method using an external standard curve. Methods.
Compared with the internal standard method, the quantitative method of the external standard curve is an accurate and reliable scientific method. Real-time fluorescence quantitative PCR using external standard curve is the most accurate and reproducible quantitative method so far. It has been recognized all over the world and is widely used in gene expression research, transgenic research, drug efficacy assessment, pathogen detection, etc. field.
Quantitative method of real-time fluorescence quantitative PCR
In Real-time PCR, there are two strategies for template quantification, relative quantification and absolute quantification.
Real-time PCR Related applications
Clinical disease diagnosis
Diagnosis and efficacy evaluation of various types of hepatitis, AIDS, bird flu, tuberculosis, STDs and other infectious diseases; eugenics and prenatal testing for thalassemia, hemophilia, gender dysplasia, mental retardation syndrome, fetal malformation; tumor marker and tumor gene detection Realize the diagnosis of tumor diseases; genetic testing realizes the diagnosis of genetic diseases.
Animal disease detection
Avian influenza, Newcastle disease, foot-and-mouth disease, swine fever, Salmonella, Escherichia coli, Actinobacillus pleuropneumoniae, parasitic diseases, etc., Bacillus anthracis.
Detection of food-derived microorganisms, food allergens, genetically modified organisms, and Enterobacter sakazakii in dairy companies.
Quantitative research on molecular biology related to medicine, agriculture and animal husbandry, and biology.
Various medical institutions at all levels, universities and research institutes, CDC, Inspection and Quarantine Bureau, veterinary stations, food companies and dairy factories, etc.
Since qPCR is a real-time quantitative detection of pathogenic pathogen gene nucleic acid, it has more unique advantages than immunological methods such as chemiluminescence, time resolution, and protein chip.