Application of Real-time PCR Technology

Real-time Q-PCR has a wide range of applications, including the study of mRNA expression, the detection of DNA copy number, and the determination of single nucleotide polymorphisms (SNPs). The following is an overview of the current applications of real-time Q-PCR in translocation gene detection, cytokine expression analysis, tumor drug resistance gene expression research, and quantitative monitoring of viral infections.

2.1 Detection of minimal residual disease

"Tumor diseases, especially hematological malignancies, are often accompanied by specific gene translocations, and this translocation can often be used as a tumor marker to monitor the effect of clinical treatment. Although the improvement of the treatment plan in the past few decades has greatly prolonged the survival period of patients, patients in remission still have the risk of relapse. Therefore, the detection of minimal residual disease (MRD) is crucial for further adjustment of the treatment plan. The application of Real-time Q-PCR is becoming an indispensable research tool for the detection of tumor micro-residue molecular markers. Quantitative determination of tumor fusion genes can guide the clinical practice of individualized treatment of patients. The most common chromosomal abnormality in acute myeloid leukemia (AML) is an reciprocal translocation t(8; 21) (q 22; q22), in which the AML-1 transcription factor gene and the MTG8 gene on chromosome 8 occur Fusion, resulting in normal AML-1 transcription regulation is affected, which may be the cause of leukemia. The current research proves that real-time Q-PCR to detect fusion genes is helpful to quantify the MRD of these patients, which is valuable as a prognostic indicator or evaluation of treatment options. The same method has also been used to quantify the level of other translocation fusion genes, such as the BCR-ABL fusion gene of chronic myelogenous leukemia (CML); the leukemia-specific TEL-AML1 fusion gene of acute lymphoblastic leukemia (ALL); Chromosomal translocation t(14;18)(q32;q21) and bcl-2 rearrangement in follicular lymphoma (FL). Many studies have benefited to a large extent from the application of real-time Q-PCR methods. With the development of technology, the application of Real-time Q-PCR will continue to expand.

2.2 Expression analysis of cytokines

Cytokines are regulatory proteins, which play a central role in the immune system by regulating the immune response (including lymphocyte activation, proliferation, differentiation, survival and apoptosis). Many different types of cells can secrete this low-molecular-weight protein, including lymphocytes, antigen-presenting cells, monocytes, endothelial cells, and fibroblasts. Cytokines can be divided into different groups; interleukin (IL-1～IL-23), interferon (IFN-α, IFN-γ, etc.), colony stimulating factor (CSF), tumor necrosis factor (TNF), tumor growth Factors (TGF-β, etc.) and chemical factors (MCP-1, MIP-1, etc.). In order to clarify the immune pathogenic pathways in many inflammatory reactions, autoimmune diseases and organ transplant rejection, reliable quantification of cytokine mRNA expression profiles is very important. Although the content of cytokines in the tested samples is often extremely low, real-time reverse PCR (RT-PCR) has become more and more popular in the quantification of cytokines due to its high sensitivity and accuracy.

2.3 Research on the expression of tumor resistance genes

Resistance to chemotherapeutic drugs is the main obstacle to the treatment of cancer patients. Since drug resistance limits the successful treatment of many tumors, it is very important to study the mechanism of tumor cells on drug resistance. The main resistance mechanisms found in the current research include: ATP-binding cassette gene superfamily (ATP-binding cassette superfamily) membrane transport protein-mediated resistance, these proteins include: MDR-1 gene encoding P-glycoprotein (P -gp), multidrug resistance-related protein (MRP), lung resistance-related protein (LRP), breast cancer resistance protein (BCRP), etc.; enzyme-mediated resistance, including topo-constructive enzyme (Topo), valley Glutathione (GSH) and glutathione-S-transferase (GST), protein kinase C (PKC), deoxycytidine kinase (deoxycytidine kinase), etc., apoptotic gene-mediated drug resistance, such as bcl -2 family, p53 gene, c-myc, etc. Multidrug resistance (MDR) is the result of multiple factors and multiple mechanisms. Real-time reverse transcription PCR (RT-PCR) is a useful means to understand tumor resistance and guide clinical treatment strategies. It can observe changes in the expression of drug resistance genes in tumor cells before and after medication and at the time of recurrence, so as to adjust the treatment plan and treatment in time. To evaluate the prognosis of the disease.

2.4 Quantitative monitoring of virus infection

The development of amplification technology has improved the ability of qualitative or quantitative detection of viruses, and has also made it possible to study the relationship between virus load and disease progression. Real-time Q-PCR is an amplification technology mainly used in the field of scientific research and diagnosis. It can not only characterize the virus, but also because of its small inter-batch and intra-batch difference and good repeatability, so it can be convenient, fast, Sensitive and accurate quantification of viral DNA or RNA sequences, and more importantly, it can dynamically study the resurrection or persistence of potential viruses throughout the course of the disease, so that clinicians and virologists can detect clinical changes, such as antiviral therapy The effect of resistance, the emergence of drug-resistant mutations, etc. At present, the most widely used method is to use Real-time Q-PCR to quantitatively determine CMV infection in patients using immunosuppressive agents in organ transfer. Studies have shown that Real-time Q-PCR detection of CMV infection in patients undergoing bone marrow transplantation is more sensitive than the traditional pp65 antigen test. Anti-CMV drug treatment can reduce the virus content in the blood. Real-time Q-PCR is useful for rapid quantification of bone CMV infection of transplant patients and monitoring of CMV resurrection is a useful tool.