Application of Fluorescence Quantitative PCR
Real-time fluorescence quantitative PCR technology refers to the method of adding fluorescent groups into 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.
Fluorescence quantitative PCR was first called TaqMan PCR, and later it was also called Real-Time PCR. So, what are the applications of fluorescence quantitative PCR?
1. Nucleic acid quantitative analysis
It is used for qualitative analysis of infectious diseases, detection of pathogenic microorganisms or virus content, such as Influenza A H1N1, detection of gene copy number of transgenic animals and plants, and detection of RNAi gene inactivation rate, etc.
2. Analysis of gene expression differences
It is used to compare the expression differences of specific genes between different processed samples (such as drug treatment, physical treatment, chemical treatment, etc.), to confirm the expression differences of specific genes in different phases and the results of cDNA chips or differential display.
3. SNP detection
The detection of single nucleotide polymorphisms is of great significance for studying the susceptibility of individuals to different diseases or the different responses of individuals to specific drugs. Due to the ingenuity of the molecular beacon structure, once the sequence information of the SNP is known, it will become simple and accurate to use this technique for high-throughput SNP detection.
4. Methylation detection
Methylation is related to many human diseases, especially cancer. Laird reported a technology called Methylight, which processes DNA before amplification so that unmethylated cytosine becomes uracil, and methylated cytosine is not affected, and specific primers and Taqman probes are used to distinguish methylated and unmethylated DNA. This method is not only convenient but also more sensitive.
5. Prenatal diagnosis
People still cannot cure genetic diseases caused by genetic material changes. So far, only prenatal monitoring can be used to reduce the birth of sick babies to prevent the occurrence of various genetic diseases, for example, in order to reduce the birth of children with X-linked genetic diseases, fetal DNA is isolated from the peripheral blood of pregnant women, and it is is a non-invasive method to use real-time fluorescent quantitative PCR to detect the Y sex determining region gene, which is easily accepted by pregnant women.
6. Pathogen detection
The fluorescent quantitative PCR detection technology can quantitatively determine pathogens such as Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, human papilloma virus, herpes simplex virus, human immunodeficiency virus, hepatitis virus, influenza virus, mycobacterium tuberculosis, Epstein-Barr virus and cytomegalovirus, etc. Compared with traditional detection methods, it has the advantages of high sensitivity, less sampling, quickness and simplicity.
7. Evaluation of drug efficacy
Quantitative analysis of hepatitis B virus (HBV) and hepatitis C virus (HCV) shows that the amount of virus is related to the efficacy of certain drugs. HCV is expressed at a high level and is not sensitive to interferon treatment, while HCV is low in titer and interferon is sensitive. During lamivudine treatment, the serum level of HBV-DNA has declined, and then if it rises again or exceeds the previous level , It indicates that the virus has mutated.
8. Tumor gene detection
Although the mechanism of tumor pathogenesis is not yet clear, it is widely accepted that mutations in related genes are the root cause of carcinogenic transformation. Increased expression and mutation of oncogenes can appear in the early stages of many tumors. Real-time fluorescence quantitative PCR can not only effectively detect gene mutations, but also accurately detect the expression of cancer genes. At present, this method has been used to detect the expression of multiple genes such as telomerase hTERT gene, chronic myelogenous leukemia WT1 gene, tumor ER gene, prostate cancer PSM gene, tumor-related viral genes and so on.