Real-time fluorescence quantitative PCR is a method for measuring the total amount of product after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction using a fluorophore. The method is used to quantify specific DNA sequences in the sample to be tested by internal or external reference methods. Since its inception, fluorescent quantitative PCR assays have become increasingly popular with laboratory teachers.

Fluorescence PCR principle: Fluorescence PCR, first called TaqManPCR and later also Real-TimePCR, is a new nucleic acid quantification technique developed by PE (PerkinElmer) in the USA in 1995. The technique is based on the addition of a fluorescently labelled probe or corresponding fluorescent dye to conventional PCR to achieve its quantitative function. The principle: as the PCR reaction proceeds, the PCR reaction products accumulate and the intensity of the fluorescent signal increases in equal proportion. With each cycle, a fluorescence intensity signal is collected so that we can monitor the change in product amount by the change in fluorescence intensity and thus obtain a fluorescence amplification curve graph.

In general, the fluorescence amplification curve can be divided into three phases: the fluorescence background signal phase, the fluorescence signal exponential amplification phase and the plateau phase. During the background signal phase, the amplified fluorescence signal is masked by the fluorescence background signal and changes in product amount cannot be determined. In the plateau phase, the amplification product no longer increases exponentially, there is no linear relationship between the end product amount and the starting template amount, and the starting DNA copy number cannot be calculated based on the final PCR product amount. Only in the exponential amplification phase of the fluorescent signal is there a linear relationship between the logarithm of the PCR product amount and the starting template amount, and we can choose to quantify this at this stage. For the convenience of quantification and comparison, two very important concepts have been introduced into the real-time fluorescent quantitative PCR technique: the fluorescence threshold and the CT value.

The threshold is an artificially set value on the fluorescence amplification curve. exponential phase of PCR amplification.

Ct value: is the number of cycles that the fluorescence signal in each reaction tube has undergone to reach the set domain value.

The relationship between the Ct value and the starting template: studies have shown that the Ct value of each template has a linear relationship with the logarithm of the starting copy number of that template, the more copies of the starting copy number, the smaller the Ct value. The Ct values are relatively stable. A standard curve can be made using a standard with a known starting copy number, where the horizontal coordinate represents the logarithm of the starting copy number and the vertical coordinate represents the Ct value as shown in the figure below.

Therefore, by obtaining the Ct value of an unknown sample, the starting copy number of that sample can be calculated from the standard curve.

The Ct value is not constant and can be influenced by different samples and different instruments, even if the same sample is repeated 2 times on the same instrument, the Ct value can vary.

Quantitative fluorescence assays: Quantitative fluorescence assays can be divided into fluorescent probes and fluorescent dyes depending on the markers used. Fluorescent probes include Beacon technology (molecular beacon technology, represented by American Tagyi), TaqMan probes (represented by ABI) and FRET technology (represented by Roche); fluorescent dyes include saturated fluorescent dyes and non-saturated fluorescent dyes, the typical representative of non-saturated fluorescent dyes is SYBRGreen I, which is commonly used now; saturated The typical representative of non-saturated fluorescent dyes is SYBRGreenⅠ; saturated fluorescent dyes are EvaGreen, LCGreen, etc.

SYBRGreenI is a commonly used DNA binding dye for fluorescent PCR, which binds non-specifically to double-stranded DNA. In its free state, SYBRGreenI emits a weak fluorescence, but once bound to double-stranded DNA, its fluorescence increases 1000-fold. Therefore, the total fluorescence signal emitted by a reaction is proportional to the amount of double-stranded DNA present and increases as the amplification product increases.

Advantages of double-stranded DNA binding dyes: simple experimental design, only 2 primers required, no need to design probes, no need to design multiple probes for rapid testing of multiple genes, ability to perform melting point curve analysis, test the specificity of the amplification reaction, low initial cost, good generality and therefore more commonly used in research at home and abroad.

Fluorescent probe method (Taqman technique): When PCR amplification is performed, a pair of primers is added along with a specific fluorescent probe. When the probe is intact, the fluorescence signal emitted by the reporter group is absorbed by the quenched group and is not detected by the PCR instrument; during PCR amplification (in the extension phase), the 5'-3' cleavage activity of the Taq enzyme degrades the probe enzymatically, making the reporter fluorescence group and quenched fluorescence group

Applications of fluorescent quantitative PCR.

Molecular biology research:

1. Quantitative nucleic acid analysis. Quantitative and qualitative analysis of infectious diseases, detection of pathogenic microorganisms or viruses, such as the recent influenza A (H1N1) epidemic, detection of gene copy numbers of transgenic plants and animals, detection of RNAi gene inactivation rates, etc.

2. Differential gene expression analysis. Comparison of gene expression differences between treated samples (e.g. drug treatment, physical treatment, chemical treatment, etc.), expression differences of specific genes in different phases and confirmation of cDNA microarray or differential expression results

3. SNP detection. The detection of single nucleotide polymorphisms is important for the study of individual susceptibility to different diseases or individual response to specific drugs, and because of the ingenious structure of molecular beacons, once the sequence information of a SNP is known, it is easy and accurate to use this technique for high-throughput SNP detection.

4. Methylation detection. Methylation is associated with many human diseases, especially cancer, and Laird reported a technique called Methylight, which treats DNA prior to amplification so that unmethylated cytosine becomes uracil and methylated cytosine is unaffected, using specific primers and Taqman probes to distinguish between methylated and unmethylated DNA. more sensitive.

Medical research:

1. Prenatal diagnosis: people cannot treat hereditary diseases caused by altered genetic material, and so far, they can only reduce the number of sick babies born through prenatal monitoring to prevent the occurrence of various hereditary diseases. This is a non-invasive method that is easily accepted by pregnant women.

2. Pathogen detection: The fluorescent quantitative PCR assay allows quantitative determination of pathogens such as gonococcus, Chlamydia trachomatis, Mycoplasma solium, human papilloma virus, herpes simplex virus, human immunodeficiency virus, hepatitis virus, influenza virus, Mycobacterium tuberculosis, EBV and cytomegalovirus. It has the advantages of high sensitivity, low sample size, rapidity and simplicity compared to traditional testing methods.

3. Drug efficacy assessment: quantitative analysis of hepatitis B virus (HBV) and hepatitis C virus (HCV) shows that the relationship between viral load and the efficacy of certain drugs. If the serum level of HBV-DNA decreases during lamivudine treatment and then increases again or exceeds the previous level, it is indicative of virus mutation.

4. Oncogenetic testing: Although the mechanism of tumour development is not yet clear, it is widely accepted that mutations in relevant genes are the underlying cause of oncogenic transformation. Increased expression and mutation of oncogenes can be seen in the early stages of many tumours. Real-time fluorescence quantitative PCR is not only effective in detecting mutations in genes, but can also accurately detect the expression of oncogenes. This method has been used to detect the expression of a variety of genes including the telomerase hTERT gene, the chronic granulocytic leukaemia WT1 gene, the oncogenic ER gene, the prostate cancer PSM gene, and tumour-associated viral genes.

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