Ask students majoring in biology to help introduce "PCR"

I. Overview of PCR

1, what is PCR?

Polymerase chain reaction (PCR) is also called cell-free molecular cloning or primer-directed enzymatic amplification of specific DNA sequences in vitro. Invented by Dr. Mullis from the genetics department of American scientist PE(Perkin Elmer Perkin-Elmer), Mullis won the 1993 Nobel Prize in Chemistry due to the epoch-making significance of PCR technology in theory and application.

2. The development history of 2.PCR technology

The principle of PCR is mediated by a pair of primers, which can rapidly enzymatically amplify specific gene (DNA) fragments in vitro of animals and plants. After n thermal cycles of amplification, the number of specific genes in the amplified product is (1+e) n (0 < e < 1), and the amplification efficiency is = times, which makes it possible to detect the specific genes in the amplified product.

Polymerase chain reaction has experienced four generations of products since 1993:

First Generation: Artificial/Robot Water Bath Gene Amplification

As shown in the above figure, three constant temperature water bath boxes are used to keep the temperature of the three water baths at three temperatures: high temperature denaturation temperature of PCR (such as 94℃), low temperature renaturation temperature (such as 58℃) and appropriate temperature extension temperature (such as 72℃). Then take a bath in water bath boxes with different temperatures by hand in a basket with PCR specimen test tubes, and the constant temperature time of specimens in each water bath box is timed by a stopwatch. In this way, PCR samples can complete the following forms of thermal cycle:

94℃30 seconds 58℃45 seconds 72℃60 seconds

40 cycles

The characteristics of this method are: the labor intensity of experimenters is high, and errors are easily caused by fatigue; The advantages are: simple equipment, less investment, no need for heating and cooling process compared with automatic gene amplification instrument, short experimental time, and the experiment is closer to ideal PCR reaction conditions. The fact shows that the experimental effect is good, but because the specimen tube is exposed to the air for a short time when it moves from one water bath box to another, if the moving speed is not fast enough, it will also cause temperature interference to the specimen and affect the results. Other disadvantages of this method include that it can only be limited to three temperature steps (some PCR reactions need more than three temperature steps), liquid pollution, and it is difficult to burn the water temperature to 94℃ denaturation temperature in low pressure area.

In order to improve the automation level of this method, a manipulator device is designed to replace the above-mentioned manual sample moving process, and a manipulator water bath gene amplification instrument is composed. This improvement solves the problem of high-intensity labor of experimenters, but it also brings high faults caused by frequent relative movement of manipulator parts in long stroke. This type of gene amplifier was sold in Beijing and Shanghai in the mid-1990s and was widely used. It has made a positive contribution to the development of molecular biology in China, and was gradually replaced by a more automatic amplifier, and now it is rarely used by units.

The second generation: automatic control qualitative gene amplification instrument

Compared with the above-mentioned water bath amplifier, some people call it dry gene amplifier, which is the most representative amplifier, including the third and fourth generations introduced later. All of them are based on the second generation and integrate quantitative detection.

Third Generation: End Point Quantitative/Semi-quantitative

The second generation of qualitative PCR can only judge negative and positive, and cannot evaluate the concentration and quantitative analysis of specific nucleic acids. Quantitative PCR can at least achieve the following functions that qualitative PCR cannot:

? Detection of latent virus concentration

? Degree of infection

? Quantitative determination of pathogenic pathogens

? Efficacy evaluation of antiviral drugs

? Detection of viral load in recovery period

Since ABI Company of America invented the first fluorescence quantitative PCR instrument in 1996, PCR technology and application have developed rapidly from qualitative to quantitative. The advantage of endpoint quantitative PCR is low equipment investment. For scientific research units and medical institutions whose domestic economic conditions can't afford expensive real-time fluorescence quantitative PCR instruments, the existing second-generation conventional qualitative PCR instruments can be used, and a special single-hole PCR terminal product fluorescence quantitative detector can be added to start working and play a quantitative role. The endpoint quantitative PCR technology is an intermediate product of the transition from qualitative to real-time quantitative. However, there are fewer imported products and more domestic instruments, such as TL988 of Xi An Tianlong and DA620 of Shanghai Lingguang.

The fourth generation: real-time quantitative PCR

QCPR-DNA/RNA real-time fluorescence quantitative detection system is composed of real-time quantitative QCPR instrument, real-time fluorescence quantitative reagent, general computer and automatic analysis software.

See the figure below: (omitted)

2. Advantages of real-time fluorescence quantitative PCR:

The equipment consists of a fluorescence quantitative system and a computer, which is used to monitor the fluorescence in the circulation process. A computer connected to a real-time device collects fluorescence data. The data are displayed in the form of charts through the developed real-time analysis software. The raw data is plotted as fluorescence intensity versus cycle times. After collecting the raw data, you can start the analysis. The software of real-time equipment can normalize the collected data to make up for the difference of background fluorescence. After standardization, a threshold level can be set, which is the level at which fluorescence data is analyzed. The number of cycles experienced by the sample when it reaches the threshold level is called Ct value (the number of cycles at the limit point). The threshold should be set to maximize the amplification efficiency in exponential period, so as to obtain the most accurate and repeatable data. If there are also standards with corresponding concentrations, linear regression analysis will produce a standard curve, which can be used to calculate the concentration of unknown samples.

3. Principle of real-time fluorescence quantitative PCR technology.

The so-called real-time Q-PCR technology refers to the method of adding fluorescent genes to the PCR reaction system, monitoring the whole PCR process in real time by using the accumulation of fluorescent signals, and finally quantitatively analyzing the unknown template through standard curves. In the development of real-time technology, two important discoveries play a key role: (1) In the early 1990s, the discovery of exonuclease activity of Taq DNA polymerase can degrade specific fluorescent probes, making it possible to indirectly detect PCR products. (2) After that, the whole reaction process was monitored in real time in a closed reaction tube by using a fluorescent double-labeled probe. The combination of these two findings and the commercial development of corresponding instruments and reagents led to the application of real-time Q-PCR in research work.

The number of copies of DNA produced during PCR reaction increased exponentially. With the increase of the number of reaction cycles, the final PCR reaction no longer exponentially generates templates, thus entering the platform period. In traditional PCR, gel electrophoresis separation and fluorescence staining are often used to detect the final amplification products of PCR reaction, so it is not reliable to quantify PCR products by this end-point method. In real-time Q-PCR, the whole amplification process of PCR reaction is monitored in real time, and the fluorescence signals related to amplification are continuously analyzed. As the reaction time goes on, the change of the monitored fluorescence signal can be plotted as a curve. In the early stage of PCR reaction, the level of fluorescence can't be clearly distinguished from the background, and then fluorescence comes into exponential period, linear period and final plateau period, so the amount of PCR products can be detected at a certain point in the exponential period of PCR reaction, and the initial content of template can be inferred from this. In order to compare the detected samples conveniently, it is necessary to set a certain fluorescence signal threshold during the exponential period of real-time Q-PCR reaction. Generally, the threshold takes the fluorescence signal of 15 cycles before PCR reaction as the fluorescence background signal, and the default setting is 65438 of the standard deviation of fluorescence signal of 3 ~ 15 cycles. If the detected fluorescence signal exceeds the threshold, it is considered as a real signal and can be used to define the threshold cycle number (Ct) of the sample. The meaning of Ct value is: the number of cycles that the fluorescence signal in each reaction tube experiences when it reaches the set threshold. The research shows that there is a linear relationship between the Ct value of each template and the logarithm of the initial copy number of the template. The more initial copies, the smaller the Ct value. The standard curve can be made by using the standard with known initial copy number, so the initial copy number of the sample can be calculated from the standard curve as long as the Ct value of the unknown sample is obtained.

4. Application of real-time fluorescence quantitative PCR technology in medical treatment.

⑴ Pathogen detection: At present, pathogens such as Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, human papillomavirus, herpes simplex virus, human immunodeficiency virus, hepatitis virus, influenza virus, Mycobacterium tuberculosis, Epstein-Barr virus and cytomegalovirus can be quantitatively detected by using fluorescence quantitative PCR detection technology. Compared with traditional detection methods, it has the advantages of high sensitivity, less sample consumption, rapidity and simplicity.

For example, the significance of tuberculosis gene diagnosis mainly lies in:

A. distinguish mycobacterium tuberculosis from other mycobacteria;

B, detecting tuberculosis drug resistance genes;

C. improve the positive detection rate of tuberculosis.

⑵ Prenatal diagnosis: Up to now, people can't treat hereditary diseases caused by genetic material changes, so they can only reduce the birth of sick babies through prenatal monitoring, thus preventing the occurrence of various hereditary diseases. For example, in order to reduce the birth of children with X-linked genetic diseases, it is a non-invasive method to isolate fetal DNA from pregnant women's peripheral blood and detect its Y sex-determining region gene by real-time fluorescence quantitative PCR, which is easy for pregnant women to accept.

⑶ Evaluation of drug efficacy: The quantitative analysis of hepatitis B virus (HBV) and hepatitis C virus (HCV) shows that the number of viruses is related to the efficacy of some drugs. High level HCV expression is insensitive to interferon therapy, while low titer HCV is sensitive to interferon therapy. During the treatment of lamivudine, the serum content of HBV-DNA once decreased, and then increased or exceeded the previous level again, suggesting that the virus has mutated. For example, the application and significance of PCR in HBV detection;

A. understand the number of hepatitis b virus in the body.

B. whether to copy.

C. whether it is contagious and how infectious it is.

D. whether it is necessary to take medicine.

E whether the abnormal changes of liver function are caused by virus.

F. judge what kind of antiviral drugs are suitable for patients.

G. judge the curative effect of drug treatment.

⑷ Detection of tumor genes: Although the pathogenesis of tumor is not clear, it has been widely accepted that the mutation of related genes is the fundamental cause of carcinogenesis and transformation. The increase and mutation of oncogene expression can appear in the early stage of many tumors. Real-time fluorescence quantitative PCR can not only effectively detect gene mutation, but also accurately detect the expression of oncogene. At present, this method has been used to detect the expression of telomerase hTERT gene, chronic myeloid leukemia WT 1 gene, tumor ER gene, prostate cancer PSM gene and tumor-associated virus gene. With the discovery of new tumor-related genes, fluorescence quantitative PCR technology will play a greater role in tumor research.

(5) Application in Eugenics:

In recent years, with the rapid development of economy and the improvement of people's living standards year by year, people pay more and more attention to the health of themselves and their families, especially the health of the next generation. In addition, due to the gradual deepening of family planning work in China, the number of only children has increased, and the physical quality of children has become the focus of attention of elders. Therefore, how to improve the quality of newborns and human genetic quality, that is, prenatal and postnatal care, has become very important. ① Avoid the birth of individuals with serious hereditary diseases and congenital diseases. ② Promote the reproduction of individuals with excellent physical strength and intelligence. Among them, avoiding the birth of individuals with serious genetic diseases and congenital diseases is the most basic content of prenatal and postnatal care. From the point of view of clinical eugenics, the specific work includes genetic examination of mother and fetus during pregnancy, trying to exclude the birth of common hereditary diseases, and checking whether the mother has some infectious diseases such as Toxoplasma gondii, rubella virus and chlamydia during pregnancy that are easy to cause fetal malformation. In the past, chromosome analysis was mainly used to detect genetic diseases, but most genetic diseases in clinic were genetic diseases rather than chromosomal diseases, so chromosome analysis could not detect them. If PCR gene amplification is combined with single strand conformation polymorphism analysis (sscp), restriction fragment length polymorphism analysis (RFCP), allele-specific oligonucleotide acid (Aso) dot hybridization or differential PCR, the mutation of a single gene can be easily detected, and the accuracy rate can reach over 95%, so this problem has been well solved. Common infectious disease pathogens that easily lead to fetal malformation are herpes simplex virus (HSV), rubella virus (RV), human cytomegalovirus (HCMV), TOX and chlamydia trachomatis (CT). In the past, it was difficult to diagnose the infection of these pathogens, mainly through culture. However, the culture method is time-consuming and expensive, and it is influenced by sampling, specimen preservation and medication, so it cannot be popularized in clinic. Because of its high sensitivity and specificity, PCR gene amplification method is very suitable for diagnosis and curative effect tracking of these diseases, and it is the most recommended detection method.

Application of 1.PCR gene amplification method in prenatal diagnosis of genetic diseases. Hereditary diseases are diseases caused by some function, defect or abnormality of the body caused by genetic changes, and the fundamental change lies in genetic material. Its types include monogenic genetic diseases, chromosomal genetic diseases and polygenic genetic diseases. The diagnosis of genetic diseases has its particularity besides asking about medical history, general physical diagnosis, general laboratory examination and understanding symptoms and signs. Pedigree analysis, chromosome and sex staining are the main basis for the diagnosis of genetic diseases, supplemented by related enzymatic analysis. With the rapid development of molecular biology and its wide application in the diagnosis of hereditary diseases, gene diagnosis technology was born, which greatly promoted the clinical diagnosis of hereditary diseases. Polymerase chain reaction (PCR) is one of the main techniques of gene diagnosis. This rapid and sensitive in vitro gene amplification technique can detect most known gene mutations, gene deletions, chromosome dislocations and so on. PCR technology is increasingly becoming one of the most effective and reliable methods to diagnose genetic diseases. Here are some examples of universal significance in China.

(1) Detection of thalassemia by PCR; Thalassemia is a hereditary chronic hemolytic anemia, which is the most common and common single-gene genetic disease in the world. In Guangdong, Guangxi, Guizhou, Sichuan and other places, the incidence rate is high, reaching 15% in Guangxi and other places. Thalassemia is due to the imbalance of globin caused by gene mutation, which reduces or even fails to synthesize peptide chains with normal structure, showing hemolytic anemia. The types of receptor genes are divided into α, β and γ, among which α and β thalassemia are the most common and harmful. Globin gene cluster is located on the short arm of human chromosome 6, and there are two repetitive genes located in α 1 and α2. The two α genes and their flanking sequences have great homology, which is prone to unequal chromosome exchange, leading to α gene deletion-α thalassemia. Partial deletion of α thalassemia gene includes partial deletion of α 1 gene, deletion of α2 gene, simultaneous deletion of α 1 and α2 gene and non-deletion thalassemia. PCR can be used to amplify α 1 and α2 genes, and detect whether these genes are deleted or mutated, so as to diagnose α thalassemia. The gene defect of β thalassemia is mainly manifested by the mutation of a single nucleotide in the gene sequence, or the deletion or insertion of a few bases, which reduces or deletes the normal β globin peptide chain synthesis. Dot hybridization between PCR products and site-specific oligonucleotide probes (Aso) can be used for diagnosis.

(2) Phenylketonuria: Phenylketonuria (PKU) is an autosomal recessive genetic disease. Because phenylpropionic acid hydroxylase is converted into tyrosine, phenylalanine and its metabolites accumulate in the body, leading to brain injury and irreversible mental retardation. PKU patients were normal at birth. Once the newborn is diagnosed as PKU, stop breastfeeding and take oral treatment with low phenylalanine for 8- 10 years, which can keep the patient's intelligence level developing normally. Because oral low phenylalanine is very expensive, it is unbearable for ordinary families. So prenatal diagnosis is the best choice to prevent children from being born. PAH is only expressed in hepatocytes, and the enzyme activity can not be analyzed by blood cells, fibroblasts, amniotic fluid and chorionic cells, but only by gene diagnosis. The gene change of PKU is not the deletion of all PAH genes, but the main form is point mutation with many mutation sites. PCR amplification, ASO, SSCP or amplified fragment length polymorphism analysis (AmpFLA) must be used for detection. These technologies are very complicated, and inspectors must be trained professionally.

(3) Hemophilia, hemophilia is a group of the most common hereditary coagulation disorders, which can be divided into two types according to different factor defects, (factor VIII and VII defects), and complex hemophilia, but it is not common. The incidence of hemophilia A is110000, and the factor VIII gene is located near the G6PD gene, with the full length of 186Kb. Hemophilia A cases caused by extensive gene rearrangement (deletion, insertion and duplication) are rare, accounting for only 5% of factor VIII deficiency, and most patients show single or few base mutations. Because the length of factor VIII gene is 186Kb, there are many mutation sites and the frequency of new mutations is high, it is impossible to detect every mutation from the clinical point of view, and several common mutation types can be detected. The main detection method is PCR combined with RELP analysis. The incidence of hemophilia B accounts for 20% of hemophilia, and its genetic changes and detection methods are similar to those of hemophilia A.

(4) Abnormal sexual development. The material basis for distinguishing men from women is sex chromosomes. In mammalian embryo development, the female phenotype does not need any adjustment, while the male phenotype is determined by many factors. The gene located on the Y chromosome directs SSS in men, which is called testicular determinant (TDF). Modern research shows that SRY(Y chromosome sex determination region) gene is TDF gene, which is located at the end of the short arm of Y chromosome. The deletion of SRY region easily leads to the development of 46XY karyotype individuals into females. Gene detection can detect the translocation and deletion of SRY genome, so as to diagnose abnormal gender development. This research exploring gender abnormality is very popular. In addition, there is another patient with abnormal 46XY karyotype, that is, testicular feminization, which is caused by the absence of androgen receptor (AR) gene, so that the target gene of androgen does not respond to androgen or the answer is incomplete. According to different conditions, there are different phenotypes. AR deficiency has a high frequency of new mutations, showing sporadic diseases and no family history. The AR gene is 90Kb long and located on the X chromosome. AR gene abnormalities are mostly base mutations of individual genes, which can be detected by PCR combined with ASO or SSCP.

(5) Fragile X syndrome, Fragile X syndrome (FRAX) is an X-linked mental retardation syndrome (XLMR) with the highest incidence. It is named because this syndrome is related to fragile sites on the X chromosome. The IQ of most FRAX men is below 50, and it tends to decrease with age. A YAC clone covering X- fragile site was isolated by artificial yeast chromosome (YAC) cloning technology, and a gene which can be expressed in human brain was obtained from this clone, named FMR- 1 (fragile X-mentai block-1). There is a CGG trinucleotide string (CGG)n at the 5' end of FMR- 1. There is polymorphism in (CGG)n in normal population, with 6-46 repeats. When the number of repetitions exceeds 52, the division of the region is unstable, resulting in a large increase in the number of repetitions. The length of the vector insertion fragment without clinical manifestations is less than 500bg, which is clinical. People regard 500bp as the dividing line between pre-mutation and total mutation. Cytogenetics was used to detect fragile sites before the diagnosis of Frax, but the experimental conditions were strict and the success rate was not high. At present, pre-mutation and total mutation can be diagnosed by molecular genetics. The CGG repeat sequence was amplified by PCR, and the length of the amplified product was detected. The amplified fragment of the mutant gene is amplified, and when somatic heterogeneity occurs, there are many amplification bands with different lengths or even a tail, or there is no amplification phenomenon because the amplified full mutant insert is too long to exceed the PCR amplification ability.

2. Application of 2.PCR gene amplification technology in "Torsch" diagnosis. In the early pregnancy (1-3 months), some pathogenic microorganisms are easy to cause fetal malformation. The most common pathogens are toxvirus, rubella virus (RV), herpes simplex virus (HSV), chlamydia trachomatis (CT) and cytomegalovirus (HCMV).

(1) PCR detection of Toxoplasma gondii showed that Toxoplasma gondii has a wide range of natural foci, and most of them are recessive infections in human body, which can show clinical manifestations when the resistance is low. The diagnosis of Toxoplasma gondii is difficult and the serological method is highly sensitive, but there are cross-false positives and it is impossible to determine the recent infection, long-term infection or short-term infection. The diagnostic methods are biopsy or animal biopsy, and the positive rate of these methods is too low to be popularized. PCR can detect 1-2 pathogen in samples with high accuracy, and it is the best method to detect Toxoplasma gondii at present. As a prenatal and postpartum examination, whole blood samples should be taken before pregnancy or early pregnancy. EDTA or heparin can be used for anticoagulation, and EDTA has the best anticoagulation effect, which can be used to detect whether TOX exists in peripheral white blood cells. Toxoplasma gondii detection has more important clinical significance for women with infertility, recurrent spontaneous abortion or raising small animals.

(2) Rubella virus infection: Rubella virus (RV) is a single positive RNA virus, and human is the only host of wind diagnosis virus. RV infection can lead to fetal malformation and affect the development of fetal immune system, so RV detection is very important in prenatal and postpartum health care. RV can be detected by direct culture and IgM antibody test. The culture method takes a long time and is often interfered by other viruses. IgM method is inaccurate, PCR method is sensitive, specific, simple and rapid, and it is one of the best detection methods for RV infection. Rubella virus proliferates in the upper respiratory tract after invading the body, causing facial symptoms and papules, and quickly spreads to the whole body. Samples are throat swabs, serum, chorionic villi or amniotic fluid of pregnant women. RV virus is RNA virus, and PCR amplification is complicated, so attention should be paid to the timing of sample collection and the accuracy of operation.

(3) Herpes simplex virus: Herpes simplex virus (HSV) is a DNA virus, which can cause inflammation of multiple organs and can be transmitted through sexual contact. The recurrence rate of HSVⅱⅱ infection is high, and 80% of infected people relapse within 12 months. After HSV infection is eliminated by the immune system, a few patients carry it for life and relapse when they are weak. PCR is sensitive to detect HSV and can directly identify HSVⅰ/ⅱ type.

(4) Chlamydia trachomatis, Chlamydia trachomatis (CT) is a sexually transmitted disease, which can not only cause trachoma, but also cause infection of life and death organs. Unlike gonorrhea (NG), syphilis and other classic sexually transmitted diseases, CT is easily transmitted through other contact routes. The survey results of pregnant women in a few areas show that the incidence rate of this population is as high as 20%-30%. In Europe, America and other countries, CT infection has surpassed gonorrhea, ranking first in sexually transmitted diseases. Chlamydia infection is often a recessive infection with no symptoms or specific symptoms, which is difficult to diagnose. In the past, the culture methods were time-consuming and lacked sensitivity and accuracy. When PCR is used in CT detection, we should pay attention to using different materials for different detection purposes. For the diagnosis of sexually transmitted diseases, we should take reproductive tract secretions (check exfoliated cells), while for early pregnancy patients, we should check their whole blood samples, and then check vaginal secretions in the third trimester or during labor to guide the cleaning of the birth canal.

(5) Human giant cell (HCMV), HCMV infection is very common, except a few primary infections with primary mononucleosis, most of them are recessive infections. HCMV can lurk in the body for a long time. When the body's immune function is low, the virus will be activated, causing serious diseases. If pregnant women are infected with HCMV during pregnancy, it will lead to premature delivery, malformation, stillbirth and neonatal giant cell inclusion disease. HCMV belongs to herpes virus family, which can be secreted from saliva, urine, cervical secretions and milk. The "gold standard" diagnostic test of HCMV is virus culture with monolayer fibroblasts, and other experimental tests include serological test and inclusion body test. PCR is a new detection method for HCMV, and the samples used for detection include blood and genital tract secretion swabs. When used for prenatal and postpartum health care, blood samples should be collected. For patients with HCMV infection in early pregnancy, amniotic fluid or other pregnancy products should be tested again, and the patients should be carefully followed up to make a comprehensive judgment and take corresponding medical measures.

The application of PCR in eugenics has great advantages, which makes the popularization and application of this technology just start. We should pay attention to the accuracy of the operation and try to avoid misdiagnosis. For infectious diseases, blood samples of pregnant women should be taken for testing first. When there is doubt or pathogen nucleic acid is detected in the blood, the pregnant women should be examined for amniotic fluid as soon as possible. Regardless of whether it is suspected that the fetus has a serious genetic susceptibility and a moderate risk of infection leading to malformation, the opinions of the parents of the fetus should be respected.

5. Some applications of real-time fluorescence quantitative PCR technology in research.

First, research and development of new drugs, drugs for human use and other drugs.

For some infectious diseases drugs, such as various viral diseases and bacterial diseases, in the process of new drug research and development, it can save a lot of manpower, time and money for new drug research and development. Compared with Elisa and other methods used before, real-time fluorescence quantitative PCR technology can quickly, accurately, quantitatively and sensitively determine the content of pathogens in blood or tissues, so it is helpful to analyze the effect of drugs and compare the efficacy, dosage and cost of different formulations.

This method can be used not only for human drugs, but also for animal drugs, and even for drug research of other economic animals and plants, so its application range in drug research is very wide.

The second is the research and development of drugs and treatments for ultra-early infection.

Real-time fluorescence quantitative PCR is used to determine the content of viral nucleic acid in blood, so it is not necessary to wait until antibodies are produced in patients. At the same time, because its sensitivity is not the same as that of Elisa, it can be determined at the early stage of virus infection, that is, when the virus content in blood is very low. Therefore, a new field has emerged, that is, how to use drugs when the virus or bacteria content is very low, what drugs to use, how much to use, etc., to contribute to the elimination of diseases in the ultra-early stage.

Three, drug efficacy research, human drugs and other drugs

For some new drugs that have been on the market or old drugs that have been used for a long time, we can further study the effect, dosage and time of medication, so as to further study the rational application of these drugs and benefit mankind. Because the methods used in the past can not be accurately quantified and the sensitivity is not enough, there are many things to be studied. On the one hand, there are many places worth studying in this field, which is also of great significance.

Fourth, study new prognostic indicators.

For infectious diseases, it is considered that drugs can be stopped after they act to a certain extent, but there is no clear indicator of when to stop taking drugs. Due to the limitation of sensitivity and accuracy of detection methods, this index may be unreasonable. Therefore, it is necessary to further study the cure index and its relationship with recurrence rate and the transformation of diseases into other diseases, so as to lay a solid theoretical foundation for the complete cure of diseases by drugs or therapies.

Verb (abbreviation for verb) develops new diagnostic and test reagents.

Many existing diagnosis and detection methods can't meet the requirements of rapidity, sensitivity and quantification at the same time, such as the existing culture method and Elisa method, but the fluorescence quantitative PCR method can do this, thus developing new reagents for clinical diseases, commodity inspection, grain and oil inspection, food inspection, blood inspection and so on. So as to improve the sensitivity, speed and accuracy of inspection. There are many kinds of such reagents, and the social and economic benefits of the developed reagents are enormous.

Six, blood test missed rate

Because real-time fluorescence quantitative PCR is much more sensitive than Elisa, blood stations or blood research institutes generally use it to study the missed detection rate of Elisa, that is, analyze the blood samples with negative Elisa, and then re-examine them with real-time fluorescence quantitative PCR. During re-examination, 24 blood samples with negative Elisa are generally mixed into one sample for testing. A case of HIV was found in the blood used in Shanghai blood products by this method, and the patient was confirmed to be HIV positive by testing the donor. Beijing Red Cross Blood Center is calculating the missed detection rate of 50,000 Elisa negative blood samples from Beijing Blood Station. Because the Elisa reagents, methods and batch numbers used in different regions are different, it is necessary to carry out this work in different regions.

Because of its rapidity, sensitivity and quantification, fluorescence quantitative PCR has many applications in research and development, such as studying the relationship between individual genotypes and drug efficacy. Researchers can also develop new uses according to their own research projects.

Product introduction of TL988 real-time fluorescence quantitative PCR detection system

1, how to choose a suitable PCR instrument?

Looking back on the development of molecular biology, the invention and popularization of PCR technology is undoubtedly one of the most important chapters. In the continuous development and innovation of PCR technology in recent 20 years, the most striking thing is real-time quantitative PCR (ORQPCR). Quantitative PCR technology has truly achieved a leap from qualitative to quantitative PCR. By monitoring the PCR process in real time, the initial template concentration can be quantified specifically, sensitively, quickly and accurately, which has been widely used in scientific research and clinical diagnosis. Based on the principle of fluorescence quantitative PCR, our company introduced the types and technologies of fluorescence quantitative PCR instrument in detail, which provided detailed reference for the selection of quantitative PCR instrument.

Quantitative PCR instrument mainly consists of two parts, one is PCR system, and the other is fluorescence detection system. From the above principle introduction, it is not difficult to see the key to choose a quantitative PCR instrument-because quantitative PCR must be quantified by comparing samples with standards. For a quantitative PCR system, the important parameters are not only the accuracy of temperature control, the speed of temperature rise and fall, etc. Traditional PCR, but also the uniformity between sample holes, to avoid small differences being multiplied. As for the fluorescence detection system, multi-color multi-channel detection is the mainstream trend today-the more excitation channels of the instrument, the more fluorescein types of the instrument, and the wider the application scope of the instrument; Multichannel means that multiple fluorescence in a sample can be detected at the same time, and the instrument can detect multiple templates or internal standard+sample in a single test tube at the same time. The more channels, the wider the scope of application and the stronger the instrument performance. The fluorescence detection system mainly includes excitation light source and detector. Excitation light sources include halogen tungsten lamp light source, argon ion laser and LED light source. The former can be equipped with multi-color filter to realize different excitation wavelengths, while monochrome LED is low in price, low in energy consumption and long in life. But because it is monochromatic, different LEDs are needed to better realize different excitation wavelengths. The monitoring system includes ultra-low temperature CCD imaging system and photomultiplier tube. The former can image multiple points at a time, while the latter has high sensitivity but can only scan one sample at a time. It takes a long time to scan and detect multiple samples one by one. Another factor that needs to be considered in quantitative PCR instrument is software design. At present, newer instruments have good supporting software to meet daily use. -With the application of quantitative PCR technology in clinical diagnosis, diseases