Analysis of the key points of fluorescence quantitative PCR
One, Principle of Fluorescence Quantitative PCR
Fluorescent quantitative PCR (Real-time PCR) refers to the addition of fluorescent groups to the PCR amplification reaction system, through the real-time detection of the fluorescent signal of each cycle product in the amplification reaction, and finally quantitative analysis of the unknown template through the standard curve Methods.
Take probe fluorescence quantitative PCR as an example: During PCR amplification, a specific fluorescent probe is added while adding a pair of primers, and both ends of the probe are labeled with a reporter fluorophore and a quencher fluorophore. At the beginning, the probe is completely bound to any single strand of DNA, the fluorescent signal emitted by the reporter group is absorbed by the quenching group, and the fluorescent signal cannot be detected; during PCR amplification, Taq enzyme digests and degrades the probe. Separate the reporter fluorophore and quencher fluorophore, so that the fluorescence monitoring system can receive the fluorescent signal, that is, every time a DNA strand is amplified, a fluorescent molecule is formed, and the accumulation of fluorescent signal is completely synchronized with the formation of PCR products .
In traditional PCR detection, staining and electrophoretic separation are required after the amplification reaction, and it can only be used for qualitative analysis and cannot be accurately quantified. It is easy to cause contamination and false positives, which limits its application. Real-time quantitative PCR technology not only realizes the quantification of templates, but also has the characteristics of high sensitivity, better specificity and reliability, high degree of automation, and pollution-free, making fluorescent quantitative PCR gradually replace conventional PCR.
In the first few cycles of the PCR amplification reaction, the fluorescence signal changes little and approaches a straight line. Such a straight line is the baseline. This line can be automatically generated or manually set. After that, the reaction will enter an exponential growth period. During this period, the amplification curve is highly reproducible. During this period, a fluorescence threshold line can be set, which can be set at any position in the exponential amplification phase of the fluorescence signal. The default setting of the threshold is 10 times the standard deviation of the fluorescence signal for 3-15 cycles. The number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold is called the CT value. This value has a linear relationship with the logarithm of the initial concentration, and the value is reproducible.
The greatest significance of the Ct value is to calculate the expression level of the target gene. At this time, there are two concepts that are easily mentioned, namely absolute quantification and relative quantification. The purpose of absolute quantification is to determine the number of molecules of the target gene in the sample. This is commonly referred to as copy number. The purpose of relative quantification is to determine the relative proportion of the content of target genes in two or more samples without knowing their copy number in each sample.
The Ct value can of course be used to calculate these two quantitative results, but the absolute quantitative experiment must use an absolute standard with a known copy number, and a standard curve must be drawn. Relative quantification can be used as a standard curve or not. The production of absolute standards is difficult and difficult to obtain, and laboratories basically choose relative quantitative methods to calculate relative gene expression.
Two, Control in real-time PCR
The purpose of the control is to monitor each link of the test, so we sort out the control according to the test process.
The process of conventional fluorescent quantitative PCR is: sample collection-transportation-sample processing-nucleic acid extraction-reverse transcription (RNA virus)-amplification-result reading.
Let's take a look at which controls can be used in each link?
1. Positive control and negative control
The positive control and the negative control refer to the extraction and amplification of a known infected sample and a known uninfected sample under the same processing conditions, and finally a positive result and a negative result are obtained. The negative and positive control emphasizes that the treatment process is consistent with the sample and has a clear expected result.
2. Amplification control
We usually say that the positive control and negative control refer to the positive control and negative control attached to the amplification kit that do not need to be extracted. A more accurate definition should be the amplification positive control and the amplification negative control. The amplification positive control contains positive amplification template, and the amplification negative control should contain negative amplification template (matrix nucleic acid). The amplification control can only monitor whether the amplification system is normal during each amplification process, but cannot monitor the sampling, extraction, and operation processes of each sample. If it is a test kit for the detection of RNA samples, the amplification positive control uses a plasmid, and the reverse transcription process cannot be monitored.
3. Internal Control，IC
Internal standard refers to a non-target sequence molecule that is amplified together with the target sequence in the same reaction tube. There are two forms of internal standard, one is to use the internal reference gene contained in the natural sample as the internal standard, and the other is the artificially added internal standard. The biggest feature of the internal standard is that it is amplified together with the target sequence, while the other controls are amplified independently.
3.1 Internal reference genes
Usually their expression in various tissues and cells is relatively constant, and it is often used as a reference when detecting changes in gene expression levels. The internal reference gene is usually a house-keeping gene because its expression level is less affected by environmental factors, and its continuous expression changes little in almost all tissues at each growth stage of an individual. Commonly used internal reference genes include GAPDH, β-actin (BETA-actin), 18sRNA, B2M, HPRT and TBP.
The advantage of the internal reference gene is that it undergoes exactly the same processing procedure as the target gene in the sample, and can monitor the entire process of sampling, transportation, nucleic acid extraction and amplification. The disadvantage is that there are certain differences in the internal reference genes in different tissues under different species and different physiological conditions. If the sample type is oral fluid, throat swab and other samples, the low amount of internal reference gene may cause the experiment to fail. If the test is an environmental sample, the internal reference gene may be completely invalid.
3.2 Manual internal standard
Add a synthetic internal standard that does not interfere with the amplification of the target sequence. At present, most of the foreign diagnostic kits directly add internal standards to the reaction solution and amplify with the template, but such internal standards cannot monitor the process of sampling, transportation and nucleic acid extraction.
There is another form of internal standard, which uses artificially synthesized pseudoviruses. The same amount of internal standard is added to each sample before nucleic acid extraction, so that the process of sample extraction to amplification can be monitored. However, because it is added to the sample manually, it is still impossible to monitor the release of intracellular bacterial viruses.
4. Nucleic acid extraction control
The internal reference gene, pseudovirus mixed matrix, and inactivated virus mixed matrix can be used to monitor the process from extraction to amplification.
5. RT control
You can use the extracted RNA reference gene, RNA pseudovirus mixed matrix, and inactivated RNA virus mixed matrix to monitor the process from reverse transcription to amplification.
The no-RT control contains all ingredients except reverse transcriptase.
6. Blank control
6.1 Blank control without template
NTC refers to a control that does not contain a template (positive or negative template). The NC of the current kit is generally water or negative buffer, so NC is equivalent to NTC. In fact, the two have different functions.
6.2 Instrument blank control
NTC contains the primer probe and the reaction solution to monitor the primer probe whether the reaction system is contaminated. For the blank control of the instrument, I usually use an empty reaction tube to monitor the instrument for non-specific fluorescence signals.
6.3 Fluorescent dye control
The most commonly used is ROX dye. Because the fluorescence signal of real-time PCR has slight differences in each sample well, specific ROX fluorescent dyes are used. The system can calibrate the fluorescence signal of each well based on the signal value of ROX fluorescent dyes.
7. Quality control (QC)
Most of the various controls used in the above links are products used by reagent manufacturers to match the kits, and some are made by laboratories, so the entire monitoring process may not be objective and independent. Therefore, it is recommended to use third-party quality control products for each test in the control setting of the laboratory, and use the national certified reference material (RM) if possible. Because the reference material has accurate measurement value and measurement traceability, the accuracy of the entire test process can be more accurately evaluated.
8. Fixed value reference products
Quantitative detection kits in medicine need to be matched with fixed value reference materials for quantitative detection of the kit.
Three, the choice of contrast
It can be seen from the above that no control is perfect. In testing, we have to choose and match flexibly according to our own needs. The author recommends adding a quality control or reference material that can monitor the extraction to amplification during each test; add an internal standard to each test sample (if the sample types are more diverse, it is recommended to use manual internal standards, if the sample type is Relatively fixed animal tissues are recommended to use internal reference genes); amplification positive control, amplification negative control, no template control and ROX control. Conditionally add clinical positive control and clinical negative control, and use nucleic acid extraction control and reverse transcription control when there is a suspected problem in extraction or reverse transcription. Use instrument blank control irregularly.
Positive control and contamination
An undeniable fact is that positive controls can increase the risk of laboratory contamination. One source of this contamination is the contamination directly from the amplification positive control. For the amplification positive control, 10,000 copies/microliter (CT value of about 25) is usually ideal, but some kit manufacturers set the amplification positive control below the CT value of 20, which is stronger than most positive samples. Such a high concentration of control brings a huge risk of contamination to the laboratory. The reason may be simply because the reagent manufacturer is worried that its positive control is unstable and degrades when stored for a long time. Another type of contamination comes from improper handling of amplified products, or unreasonable laboratory design layout.
Initial screening and diagnosis
The relationship between the control and the purpose of the experiment seems to have never been mentioned. Based on the author’s many years of experience in testing laboratories, I share a little personal experience. I welcome your criticism and correction.
For the preliminary screening experiment, we hope to increase the true positive detection rate, that is, to improve the diagnostic sensitivity. We need the detection system to achieve the highest detection efficiency, so we must add positive controls in different links to ensure the highest detection efficiency in each link.
For confirmatory tests, we hope to increase the true negative detection rate, that is, to improve the diagnostic specificity. We need to ensure that the detection system does not appear false positives, so we need to add negative controls at different links to reduce unnecessary positive controls, and even randomly insert several negative controls in different positions of the sample tray to ensure that there is no contamination during the experiment.
Fourth, the application of fluorescence quantitative PCR
Molecular Biology Research
1 Nucleic acid quantitative analysis Quantitative and qualitative analysis of infectious diseases, detection of pathogenic microorganisms or virus content, such as the recent epidemic of H1N1 influenza, detection of gene copy number of genetically modified animals and plants, detection of RNAi gene inactivation rate, etc.
2 Gene expression difference analysis Compare the expression differences of specific genes between different processed samples (such as drug treatment, physical treatment, chemical treatment, etc.), the expression differences of specific genes in different phases, and the confirmation of the cDNA chip or difference display results
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 ingenious structure of molecular beacons, once the sequence information of the SNP is known Yes, high-throughput SNP detection using this technology will become simple and accurate.
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 However, the methylated cytosine is not affected. Using specific primers and Taqman probes to distinguish between methylated and unmethylated DNA is not only convenient but also more sensitive.
Medical Research
5 Prenatal diagnosis People cannot treat genetic diseases caused by genetic material changes. So far, only prenatal monitoring can be used to reduce the birth of sick babies and prevent the occurrence of various genetic diseases. To reduce the birth of children with X-linked genetic diseases, to isolate fetal DNA from the peripheral blood of pregnant women, and use real-time fluorescent quantitative PCR to detect the Y sex determining region gene is a non-invasive method that is easily accepted by pregnant women.
6 Pathogen detection using fluorescent quantitative PCR detection technology can detect Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, human papillomavirus, herpes simplex virus, human immunodeficiency virus, hepatitis virus, influenza virus, Mycobacterium tuberculosis, Epstein-Barr virus and giant Pathogens such as cell viruses are quantitatively determined. 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 high levels and is not sensitive to interferon treatment, while HCV is low in titers 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 mutations of oncogenes can appear in the early stages of many tumors. Real-time fluorescent 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.