Quantitative PCR is a nucleic acid quantitative technology developed on the basis of qualitative PCR technology. Real-time fluorescence quantitative PCR technology was introduced by Applied Biosystems in 1996. It is a kind of adding a fluorophore to the PCR reaction system. It uses real-time detection of the accumulation of fluorescent signals to monitor the entire PCR process. Finally, the standard curve is used to detect the unknown template. Method for quantitative analysis. This technology not only realizes the quantification of DNA / RNA template, but also has the characteristics of high sensitivity and specificity, multiple reactions, high degree of automation, no pollution, real-time and accuracy, etc. It has been widely used in molecular biology research and medicine Research and other fields. Borderless sunglasses are the most popular structure sunglasses of the year. They are assembled directly without borders, reducing the weight of sunglasses and making them lightweight and comfortable to wear. Add a sense of beauty. The lenses are colourful and colourful. It is the choice of fashion trend, frameless design, simple and generous without losing individuality. There are various colors to choose from and bring you a colorful summer. The summer is no longer dull. Free to express yourself with your nature, and express yourself bravely, and wear your tune. Rimless Sunglasses,Rose Gold Rimless Sunglasses,Reflective Rimless Sunglasses,Police Rimless Sunglasses China Guangdong Shenzhen city Omelle glasses Co., Ltd. , https://www.omelleoptical.com
1. Molecular biology research â‘´ Gene expression research: cytokine is a regulatory protein, which has an important regulatory effect on immune response and inflammatory response, including the regulation of lymphocyte activation, proliferation, differentiation, survival and apoptosis. These low-molecular proteins are secreted by lymphocytes, monocytes and endothelial cells, and the changes in their amounts are often closely related to diseases such as inflammation, autoimmune diseases, and transplant rejection. Since the tissue samples obtained are often too small to meet the detection at the protein level, in addition, the sensitivity of the commonly used protein detection method is difficult to complete the detection of very small amounts of protein products, so the use of PCR to detect the expression level of cytokine mRNA It seems very meaningful. Experimental studies have shown that quantifying the expression of cancer antigen mRNA in colorectal cancer lymph nodes can be used as an important basis for diagnosing cancer micrometastasis.
⑵Single nucleotide polymorphism (SNP) and mutation analysis: An attractive application prospect of real-time fluorescence quantitative PCR is to detect gene mutations and genome instability. Gene mutation detection is based on two probes, one probe spans the mutation site, the other is the anchor probe, hybridizes with the target sequence without mutation site, and the two probes are labeled with two different luminescent groups . If there is no mutation in the target sequence, probe hybridization will reduce the stability of the hybrid, thereby reducing its melting temperature (Tm). This allows analysis of mutations and polymorphisms. Lamivudine is an antiviral drug that is effective in the treatment of chronic hepatitis B, but there have been reports of viral mutations in clinical applications. These mutations may be the reason for treatment failure or the virus's non-response to treatment. Real-time quantitative PCR can monitor whether hepatitis B patients produce drug-resistant mutant viruses after taking lamivudine.
2. Medical research â‘´ Pathogen detection: At present, the use of fluorescent quantitative PCR detection technology can be divided into gonococcus, chlamydia trachomatis, ureaplasma urealyticum, human papilloma virus, herpes simplex virus, human immunodeficiency virus, hepatitis virus, influenza virus, tuberculosis Pathogens such as mycobacterium, EB virus, and cytomegalovirus were quantitatively determined. Compared with the traditional detection method, it has the advantages of high sensitivity, less sampling, fast and simple.
⑵ Prenatal diagnosis: So far, people can not treat genetic diseases caused by genetic material changes, only through prenatal monitoring to reduce the birth of sick infants to prevent the occurrence of various genetic diseases, such as to reduce X In the birth of children with linked genetic diseases, it is a non-invasive method to isolate fetal DNA from the peripheral blood of pregnant women and detect the gene of Y sex determination region by real-time fluorescent quantitative PCR, which is easily accepted by pregnant women.
⑶ 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. High levels of HCV expression, insensitive to interferon treatment, and low HCV titers, sensitive to interferon; during the treatment of lamivudine, the serum level of HBV-DNA had decreased, and then if it rose again or exceeded the previous level , It indicates that the virus has mutated.
â‘· Tumor gene detection: Although the mechanism of tumor onset is not yet clear, the mutation of related genes is the basic cause of carcinogenicity transformation has been widely accepted. Increased expression and mutation of oncogenes can occur early in many tumors. Real-time quantitative PCR can not only effectively detect gene mutations, but also accurately detect the expression of oncogenes. At present, this method has been used to detect the expression of various genes such as telomerase hTERT gene, chronic myelogenous leukemia WT1 gene, tumor ER gene, prostate cancer PSM gene, tumor-associated virus gene and so on. With the continuous discovery of new genes related to tumors, fluorescent quantitative PCR technology will play a greater role in the study of tumors.
Clinical significance
1. Application and significance of PCR technology in the detection of tuberculosis bacteria The significance of gene diagnosis of tuberculosis bacteria is mainly manifested in: a. Distinguishing TB from other mycobacteria; b. Detecting TB resistance genes; c. Increasing the positive detection rate of TB .
2. Application and significance of PCR technology in HBV detection
1. Understand the amount of hepatitis B virus in the body.
2. Whether to copy.
3. Whether it is contagious and how contagious it is.
4. Is it necessary to take medicine.
5. Whether abnormal liver function changes are caused by viruses.
6. Determine which type of antiviral drugs the patient is suitable for.
7. Judge the efficacy of drug treatment.
3. Application and significance of PCR technology in HCV detection
HCV is the main pathogenic factor causing hepatitis after blood transfusion. Because its content in blood is very low, it is only 1% of HBV. Its immunological marker is only anti-HCV, and the virus isolation has not been successful so far. Detection is much more difficult than HBV, therefore, PCR technology is particularly important in the detection of HCV, and its significance is mainly manifested in:
3.1 Anti-HCV can be used as an indicator for the diagnosis of HCV infection. Due to differences in immune function among individuals, some patients have anti-HCV late, and those with immunocompromised or immunosuppressed therapy may not even produce anti-HCV; therefore, HCV-RNA is a definitive marker of HCV infection. According to reports, the detection rates of ELISA anti-HCV in patients 30-30 days and 6-30 were 45% and 67%, indicating that ELISA detected anti-HCV has a relatively high miss rate.
3.2 No anti-HCV is produced during the "window period", but RNA can be detected.
3.3 HCV-RNA quantification can guide the use of drugs and provide objective indicators for the observation of efficacy and prognosis. High serum HCV copies are insensitive to interferon and low sensitive. According to reports, HCV <100pg / ml group: 11/30 turn negative HCV> 100pg / ml group: 1/30 turn negative.
HCV-RNA continued to maintain high levels may have a poor prognosis.
3.4 Anti-HCV positive does not substitute for symptomatic infection. The diagnostic criteria for hepatitis C is HCV-RNA (+).
4. Application of PCR in CT detection
CT is one of the main pathogenic microorganisms of nongonococcal urethritis, and sometimes can cause infertility and other diseases. CT is obligate cell parasitism. Generally, it cannot grow in culture, and can only replicate and reproduce in live cells. The laboratory observes the inclusion bodies through Mcloy and Hela-229 cell culture, which can be regarded as a diagnostic "gold standard method". However, due to the complicated operation, high technical requirements and long time, it is not suitable for clinical detection. Immunological methods include direct immunofluorescence, EIA, etc., but both of these methods are less than 50% in the detection of antigen or antibody positive rate. The use of monoclonal antibodies will lose sites, and multiple antibodies will cross-react, screening in high-risk groups Zhongchang often cross-reacts with Staphylococcus aureus, Streptococcus, Neisseria gonorrhoeae, etc. to affect its specificity. Therefore, experts at home and abroad agree that the genetic diagnosis method of CT should be set as the "gold standard".
5. HAV is the pathogen of viral hepatitis A. Hepatitis A virus (HAV) is a hepadnavirus. Anti-HAV IgM and anti-HAV IgG and other antibody components are their serum markers. [Clinical Significance] Anti-HAV IgM antibodies appear in the blood within 1 to 2 weeks after the onset, and the titer drops after 3 months, and it is not easy to detect after 6 months. If the antibody is positive, acute hepatitis A can be diagnosed . Anti-HAV IgG appears a little later than anti-HAV IgM and generally exists for life. It is a protective antibody that can be used for the epidemiological investigation of hepatitis A.
6. Hepatitis D virus (HDV) is an RNA virus. Serum markers for clinical diagnosis of HDV are HDV Ag, anti-HDV IgM and anti-HDV IgG. The results of healthy people were all negative. When HDV is infected, HDV Ag is positive. Anti-HDV IgM positive, seen in acute HDV infection. Anti-HDV IgG positive is a reliable serological indicator for the diagnosis of chronic hepatitis D.
7. Hepatitis E virus (HEV) is an RNA virus. The serum markers for clinical diagnosis of HEV are anti-HEV IgM and anti-HEV IgG. The results of healthy people were all negative. Anti-HEV IgM positive reaction can diagnose acute hepatitis E, lasting 2 to 3 months. Anti-HEV IgG can be detected in the serum of patients in the recovery period, and the duration is about 1 year.
8. Evaluate the significance of fluorescence quantitative PCR (QPCR) technology for the detection of Chlamydia trachomatis and Ureaplasma urealyticum in infertile women. Method: Using QPCR technology to 60 infertility patients (infertility group) and 78 normal fertility patients (control group ) Cervical chlamydia trachomatis (CT) and Ureaplasma urealyticum (UU) were tested. Results: The positive detection rate of cervical secretion CTDNA, UUDNA and mixed infection in the infertile group was significantly higher than that in the control group (P ＜ 0.05 =. The detection of cervical CTDNA and UUDNA in pregnant patients is intentionally strong, rapid and sensitive, and has high clinical application value.
9. Detection of HPVDNA in peripheral blood of patients with condyloma acuminatum Condyloma acuminatum (CA) has a stubborn condition and is prone to relapse. Past research has always considered HPV to be a strict pro-epithelial virus. However, some studies have found that HPVDNA can be detected in the blood. Researchers from the Department of Dermatology of the First Affiliated Hospital of China Medical University speculate that condyloma acuminatum may have viremia in the course of HPV infection, which may be the source of virus for CA recurrence in susceptible sites or CA lesions in other sites. To this end, the researchers tested HPVDNA in the peripheral blood of 30 CA patients. Methods: Polymerase chain reaction (PCR) was used to detect HPV DNA in skin lesions and peripheral blood of 30 CA patients, and peripheral blood of 20 normal subjects was used as a control. Restriction enzymes Rsaâ… and Pstâ… were used to identify HPV. A PCR product with positive skin lesions and peripheral blood was selected for sequencing. Results: HPVDNA was detected in 30 patients with CA, HPVDNA was not detected in plasma, HPVDNA was detected in 8 peripheral blood mononuclear cells (PBMC) (positive rate was 26.7%), and 20 normal controls were negative . The positive rate of the two groups was corrected by χ2 test χ2 = 4.52, P <0.05, the difference was significant. The results of enzyme digestion and sequencing showed that the type of HPV in PBMC was consistent with the type of skin lesions. Conclusion: CA patients may have viremia during the course of HPV infection, which may be one of the reasons that CA is prone to relapse. The results of this study suggest that a large amount of virus multiplying locally may damage the local basement membrane band and enter the blood through the dermal blood vessels. In addition, the detection of HPV in PBMC suggests that the disease may be spread by blood other than sexual transmission.
10. Rapid diagnosis After smearing cervical mucosa, skin, oral cavity, cornea and other tissue cells, specific antibodies are used for indirect immunofluorescence or immunohistochemical staining to detect viral antigens; Wright-Giemsa staining microscopy, if nuclear inclusion is found The body and multinucleated giant cells can be considered for HSV infection; negative staining of herpes blister fluid by electron microscopy can quickly diagnose the diagnosis. In situ nucleic acid hybridization and PCR methods can be used to detect HSV DNA. The Southernblot method was used for the specific identification of PCR products, and the CSF PCR amplification was considered to be the best method for diagnosing herpes encephalitis. In addition, DNA digestion patterns can also be used for HSV identification and type analysis. HSV antibody measurement is of little significance for clinical diagnosis and is only used for epidemiological investigations.
11. Syphilis (TP), meaning: high sensitivity and specificity. Generally used for confirmation test. Can not be used to observe the effect: even if the patient undergoes sufficient anti-ume therapy, the serological response can still remain positive, even for life.
12. Objective To explore the value of polymerase chain reaction (PCR) reverse membrane hybridization technology in detecting Mycobacterium tuberculosis (TB) DNA in clinical specimens. Methods PCR reverse membrane hybridization technology was used to detect 522 tuberculosis and 60 non-tuberculosis clinical specimens TBDNA in the culture, using culture, acid-fast staining smear and conventional PCR method as a control. The positive rate of PCR reverse membrane hybridization was 80.6% (411/690), culture was 18.1% (125/690), microscopic examination 13.9% (96/690), conventional PCR is 59.6% (411/690), PCR reverse membrane hybridization method compared with conventional method (P <0.001 =, the difference is significant. PCR reverse membrane hybridization method was positive The rate is higher than conventional PCR (P> 0.05), but the difference is not significant; the false positive rate of this technique is zero. Conclusion PCR reverse membrane hybridization technique for detecting TB-DNA in clinical specimens has rapid, high specificity and sensitivity, which can be The early clinical diagnosis of tuberculosis provides a new etiological diagnosis method.
13. In recent years, polymerase chain reaction (PCR) technology has been used for the diagnosis of Hp. The sensitivity of this technology is increased by 100 times compared with oligonucleotide probes, and as few as 100 Hp can be detected. At present, the partial sequences of the Hp urease gene and 16SrRNA gene have been detected, which provides a basis for the selection of PCR-specific primers. Using a pair of oligonucleotides complementary to the 16SrRNA gene as primers (CP1 / CP2), we established a PCR technique to detect Hp infection from gastric mucosa biopsy specimens. Using this method to detect Hp standard strains NCTC 11637, NCTC11848 and 50 strains of Hp strains isolated from the gastric mucosa biopsies of clinical patients all produced a 450 bp fragment with a sensitivity of 0.1 pg of HpDNA, which is equivalent to 100 bacterial cells. 12 non-Hp strains (including Staphylococcus aureus, Staphylococcus epidermidis, Pneumococcus, Escherichia coli, Pseudomonas aeruginosa, Proteus, Shigella, and Campylobacter jejuni NCTC11351) and human gastric mucosal cells without Hp infection were negative . We also used PCR to detect 96 patients who underwent gastroscopy due to upper gastrointestinal symptoms. The results showed that the Hp positive rate of PCR examination was higher than that of conventional examination. We also tested 17 cases of Hp eradicated by conventional methods (all negative for urease test, silver staining, and culture) after treatment with different regimens, and found 4 cases of Hp positive by PCR. In the past, we mistakenly thought that Hp was eradicated and gave up treatment to some patients who failed to be detected by conventional methods. In these patients, the recurrence of Hp in the short term is actually incomplete treatment, which is very useful for guiding the treatment of Hp infection. Important clinical significance.
14. HCMV infection is one of the common complications after organ transplant recipients. HCMV infection is a systemic infection with diverse clinical manifestations. It is very similar to the clinical symptoms of transplant rejection and is easy to confuse, but the conditions for the two are diametrically opposed and completely different in treatment. To understand whether the clinical symptoms of recipients after organ transplantation are caused by HCMV infection, it is important to diagnose active HCMV infection in a timely manner. HCMV infection is an important factor affecting the success of organ or tissue transplantation. Especially in bone marrow transplant infections, if not diagnosed and treated in time, it can often cause fatal danger and transplant failure. Therefore, early diagnosis and timely and effective preventive treatment are the key to reducing the incidence and mortality of HCMV after transplantation. The detection of HCMV DNA by fluorescence quantitative PCR technology has clinical application value for the detection of infection, because the appearance of clinical symptoms or serological evidence of viral DNA before viral infection in body fluids can be used as an early indicator of HCMV infection. Treatment and prevention are of great significance. Because HCMV can be transmitted through intra-placental infection and birth canal infection, and the mortality rate of infected neonates is high, the use of PCR for early diagnosis and timely treatment measures is also important for eugenics. At present, antiviral drugs are mainly used for the treatment and prevention of HCMV infection diseases. Obviously, the efficacy of antiviral drugs requires effective observation and evaluation methods. Quantitative analysis of HCMV DNA by fluorescence quantitative PCR can detect the stability of the indicators. Through dynamic observation of the level of DNAemia to monitor the occurrence, development and prognosis of HCMV disease, timely adjust the dosage of immunosuppressants, guide antiviral treatment, and observe the efficacy , To analyze whether there is drug resistance, etc., so it can be used as a powerful means to evaluate the efficacy of various antiviral drugs.
Application of PCR technology in eugenics
In recent years, the economy has developed rapidly, and people ’s living standards have improved year by year. More and more attention has been paid to the health of themselves and their families, especially the next generation. In addition, as domestic family planning work is gradually deepening, the only child is relatively high, and the physical quality of the child has become the focus of elders. Therefore, how to improve the quality of newborns and improve human genetic quality, that is, eugenics and euthanasia has become very important. The eugenic and eugenic individual is born. ①Avoid birth of individuals with serious genetic diseases and congenital diseases. ②Promote 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 eugenics. Analyze the specific work from the perspective of clinical eugenics, including performing genetic tests on the mother and fetus during the mother ’s pregnancy to try to exclude the birth of individuals with common genetic diseases, and checking whether the mother has certain infections that are likely to cause fetal malformations during pregnancy Sexual diseases such as Toxoplasma gondii, Rubella virus, Chlamydia and other infections. In the past, the detection of genetic diseases mainly used chromosome analysis, and most of the clinical genetic diseases are genetic diseases rather than chromosomal diseases, and the chromosome analysis method cannot be detected. If PCR gene amplification method combined with single-strand configuration polymorphism analysis (sscp), restriction fragment length polymorphism analysis (RFCP) allele-specific oligonucleotide (Aso) point hybridization or differential PCR can be very It is convenient to detect the mutation of a single gene and its accuracy can reach more than 95%, so this problem has been better solved. Common infectious disease pathogens that are prone to fetal malformations include herpes simplex virus (HSVⅡ), rubella virus (RV), human cytomegalovirus (HCMV), toxoplasma (TOX) and chlamydia trachomatis (CT). In the past, the diagnosis of these pathogen infections was difficult, and the diagnosis was mainly based on the culture method. The culture method was time-consuming and expensive, and was affected by factors such as sampling, sample preservation and medication, and could not be promoted in clinical practice. PCR gene amplification method is very suitable for the diagnosis and tracking of these diseases because of its high sensitivity and high specificity. It is the most recommended test method.
1. The application of PCR gene amplification method in the prenatal diagnosis of genetic diseases. Genetic diseases are diseases caused by a certain function or defect or abnormality of the body due to changes in genetic material. The fundamental change lies in genetic material. Its types include single-gene inherited diseases, chromosomal inherited diseases and polygenic inherited diseases. The diagnosis of genetic diseases is special in addition to asking about medical history, general physical diagnosis, general laboratory examination, and understanding of symptoms and signs. In the past, genealogy analysis, chromosome and sex chromaticity test were used as the main basis for the diagnosis of genetic diseases, and then supplemented by related enzyme analysis to make a diagnosis. With the rapid development of molecular biology and its extensive application in the diagnosis of genetic diseases, gene diagnosis technology was born, and gene diagnosis technology has greatly promoted the clinical diagnosis of genetic diseases. Polymerase chain reaction (PCR) technology is one of the main techniques of gene diagnosis. This rapid and sensitive gene in vitro amplification technology combined with a variety of molecular biology methods can detect most of the known gene mutations, gene deletions, chromosomal dislocations, etc. PCR technology is increasingly becoming the most effective and most effective in the diagnosis of genetic diseases One of the reliable methods. Here are a few examples of general significance in China.
, Guizhou, Sichuan and other places have a higher incidence, up to 15% in Guangxi and other places. Thalassemia is due to the imbalance of globin due to genetic mutations, which reduces the amount of normally-structured peptide chain synthesis or even the synthesis of hemolytic anemia. Accepted gene types are divided into α, β, γ, etc. Among them, α and β thalassaemia are the most common and most harmful. The globin gene cluster is located on the short arm of human No. 6 dye. There are two duplicate gene genes located in α1, α2, two α genes and their flanking sequences have great homology, prone to unequal exchange of chromosomes, resulting in α Gene deletion-alpha thalassaemia. The partial deletion of α-thalassemia gene includes partial deletion of α1 gene, deletion of α2 gene, simultaneous deletion of α1 and α2 genes, and non-deletion type thalassemia. PCR technology can be used to amplify the α1 and α2 genes to detect whether these genes are missing or mutated to diagnose α-thalassemia. The β-thalassemia gene defect mainly manifests as a mutation of a single nucleotide in the gene sequence, or the deletion or insertion of a few bases, so that the normal β-globin peptide chain synthesis is reduced or deleted. It can be diagnosed by spot-specific hybridization of PCR products using site-specific oligonucleotide probes (Aso).