Molecular diagnosis uses DNA~RNA or protein molecules as diagnostic materials to check the existence of human endogenous genes or exogenous (pathogen) genes~ Defects or abnormal expressions are methods and processes for specific diagnosis of human conditions or diseases . With the implementation of the Human Genome Project and the Post Genome (Proteome) Project, molecular diagnostics has been further improved and matured. The combination of molecular biology and modern medicine has made laboratory diagnosis far beyond the scope of purely assisted clinical diagnosis. It is playing more and more in the fields of disease prevention ~ prognosis judgment and efficacy monitoring ~ health status evaluation and disease prediction. The greater the role, it is no exaggeration to say: Molecular diagnosis is an epoch-making detection method that will have a significant impact on the development of medicine. Molecular diagnosis has now become an important indicator to measure the overall medical level of a country and region. At this time, it is very important to think carefully ~ analyze and summarize the basic strategies of molecular diagnosis. Grasping the strategy of molecular diagnostics is conducive to consolidating the current research results of molecular diagnostics, and is also conducive to further exerting the role of molecular diagnostics in clinical medicine. It is also conducive to clarifying the development direction of molecular diagnostics.

Medical diagnostics is the science of studying the basic principles and methods of diagnosing diseases as a new branch of laboratory diagnostics. Molecular diagnostics should always follow the basic principles of laboratory diagnostics. That is, in the laboratory, the test materials (including DNA~RNA and protein) of the human body are tested and analyzed to identify the cause of the disease~the pathogen~the condition, etc.~identification and interpretation for the clinician to accurately verify the clinical impression diagnosis~analysis of the condition ~ Explore the occurrence and development of diseases to provide objective evidence. Although molecular diagnosis is also used in the detection of drug resistance ~ curative effect monitoring ~ health and epidemic prevention ~ health check ~ disease prediction and individualized treatment, molecular diagnosis still needs to follow the basic principles of laboratory diagnostics. The development of molecular diagnostic technology contains huge business opportunities. Therefore, in order to carry out research and application of molecular diagnostics safely, effectively and legally, patent laws should also be actively implemented.

Adopt different molecular diagnostic strategies and methods for different types of diseases and their possible causes

Infectious diseases are caused by pathogens such as bacteria ~ viruses ~ mycoplasma ~ chlamydia ~ parasites invading the body. In the past, these pathogens were mostly detected by microbiology ~ immunology and hematology related methods, but these methods are limited by sensitivity and specificity. Difficulty in early diagnosis G With the elucidation of the gene structure of various pathogens, it is possible to detect the infectious pathogen itself (RNA or DNA) specifically using molecular diagnostic technology early ~ fast ~ sensitive ~. The application of molecular technology to directly determine the genes of these pathogens can not only confirm the diagnosis of microbial infections, but also diagnose carriers (viruses) or latent infections, and can also classify infectious pathogens and monitor drug resistance. Therefore, the molecular diagnostic test objects for infectious diseases mainly include infectious viruses ~ bacteria ~ parasites ~ mycoplasma ~ chlamydia and other exogenous biological DNA or RNAG that exist in the human body. In principle, this type of detection is generally based on known foreign sources. DNA or RNA sequence can be diagnosed quickly, simply and accurately by PCR or RT-PCR. Therefore, the genetic diagnosis of this type of disease is extremely applicable and can be widely used in clinical practice under the premise of strict quality control. The diagnosis strategy can be divided into the following two types: One is the general detection strategy, which only needs to provide whether there is a certain pathogen infection; the second is the complete detection strategy, which is not only to diagnose the pathogen but also to type (including subtypes). Type) and drug resistance. All hereditary diseases are related to mutations of one or more genes. There are two strategies for molecular diagnosis of hereditary diseases: 1. Direct diagnosis strategy (DNA sequence analysis of the gene mutation of the disease. Directly reveal the cause of heredity A variety of genetic defects that occur in the disease; 2, 2, indirect diagnosis strategies (except for the DNA sequence analysis of the disease gene mutations. Other diagnostic methods. That is to determine the chromosome with the genetic defect in the proband ~ the allelic type and related gene Haploid, etc., then look for other members of the family to find out whether the subject also has this chromosome ~ allelic and haplotype of related genes, etc. In addition, prenatal genes (direct or indirect. Diagnosis) are carried out based on genetic disease risk assessment. It is a good way to prevent genetic diseases ~ control population quality. There are two reasons for malignant tumors, endogenous and exogenous. Endogenous factors are changes in the internal structure and function of the body, such as genetic ~ immune deficiency ~ metabolic abnormalities Etc.; Exogenous factors refer to various carcinogenic factors in nature, that is, environmental factors. The occurrence and development of tumors are the result of multi-factor mutual cooperation, and the abnormal gene level is the key reason. Therefore, in this sense, tumors are a kind of genes. At present, the molecular diagnosis of tumors can adopt three strategies: 1. Detection of tumor-related genes; 2. Detection of tumor-related virus genes; 3. Detection of tumor markers, essentially providing tumor-related gene information can be classified as direct Diagnosis and detection of tumor-associated virus genes or tumor markers are mainly indirect diagnosis.

Carry out different levels of molecular diagnosis according to the purpose of genetic testing

The basic strategy of molecular diagnosis is closely related to the cause of the disease. The causes of human diseases include internal and external factors: internal factors mainly refer to genetic factors such as gene structure ~ changes in gene expression status. The changes in gene structure include point mutations ~ insertions ~ deletions ~ rearrangements ~ translocations ~ genetic structural polymorphisms Mutation ~ pre-virus insertion, etc.; external factors refer to external environmental factors such as lifestyle ~ work environment ~ mental conditions and the invasion of various infectious pathogens. According to the purpose of genetic diagnosis, there are five basic methods to choose from.

Chromosome analysis

For the detection of large fragments of DNA, in addition to traditional cytogenetics techniques, more advanced chromosome analysis techniques such as fluorescence in situ hybridization FISH can be used to use cosmid ~ bacterial artificial chromosomes BACD or yeast Artificial chromosomes (yeast artificial chromosomes YAC) can be used as probes to obtain very stable hybridization effects. Comparative genomic hybridization (CGH) technology can partially compensate for the shortcomings of the above-mentioned technology and overcome the information it provides is limited to the probe coverage. area.

DNA content determination

DNA content determination is also a part of DNA analysis. G cells that carry a variety of genes (including genes that regulate growth and apoptosis) often contain abnormal amounts of nuclear DNA, which can be detected by flow cytometry. Changes in DNA content (DNA index) relative to normal cells may indicate the increase or loss of a certain chromosome (aneuploidy) or the presence of an extra set of chromosomes (polyploidy).
In addition, the cellular DNA content profile of the tissue can reflect the ratio of cell cycle S to G2 phase cells. In some tumors (such as breast cancer), if the ratio is high, it indicates a poor prognosis.

Gene mutation detection

If a certain mutation in a disease-causing gene has a direct causal relationship with the disease, it is ideal to detect this gene mutation as a diagnostic basis. Fully or partial understanding of disease-causing genes, complete or partial understanding of molecular mechanisms, but well-established rules, direct detection and analysis of mutations is the most powerful and accurate method of molecular diagnosis. Since a gene can have different mutation types, it is necessary to develop a reasonable and convenient strategy for the tested individual. Generally, the deletion of large DNA fragments often involves multiple genes, resulting in a mixture of multiple clinical phenotypes. Therefore, in addition to chromosome analysis, Southern blot analysis or PCR detection is usually used for larger deletions. Insertion and deletion of small fragments~ The detection of point mutations is mainly because the disease-causing gene can produce mutations at any site, so that the same disease has different mutation types. Due to the emergence of PCR technology, the use of known gene sequences to design primers to directly detect the observed object The presence or absence of has become very convenient. The use of PCR combined with single-stranded conformational polymorphism analysis method (PCRSSCPD can sensitively detect single point mutations. Although gene mutation detection has direct advantages, because most pathogenic genes have not been cloned, some pathogenic genes are known Its approximate location on the chromosome, but the exact gene structure and molecular mechanism are not known. Therefore, mutation detection methods cannot be used for many diseases.

Gene linkage analysis

Because adjacent genes on the same chromosome are inherited together, there is a linkage relationship with each other. Linkage analysis should be performed on disease-causing genes that have not been identified but are at least located in specific chromosomal regions. As long as the existence of related genes linked to the pathogenic gene is identified, it can be determined whether the subject has the pathogenic gene. The method of molecular polymorphism marker tracking can be used to isolate mutant alleles in a family. This type of analysis involves not only the collection of DNA samples from the proband but also the DNA samples of affected and unaffected family members in two to three generations. In order to be able to reduce the erroneous results due to exchange to the minimum, the polymorphic marker must be close enough to the disease-causing gene (or within the gene. Classical linkage analysis mainly uses restriction fragment length polymorphism (RFLP) as genetic markers. Family analysis. In recent years, STR, as a DNA polymorphic marker, has overcome many shortcomings of RFLP for linkage analysis, and has achieved rapid development in linkage analysis. It has gradually replaced RFLP. Facts show that the rate of false diagnosis caused by chromosome recombination rate except for individual cases Less than 1%. Therefore, the linkage analysis of DNA markers is usually reliable. In order to ensure the correctness of the genetic diagnosis of linkage analysis, it is necessary to look for 2~3 genetic markers on both sides of the detected gene or locus so that it can be prevented by haplotype analysis. Deviations due to reorganization.

Detection of gene expression status

Molecular diagnosis can detect not only the structural abnormality of genes but also the gene expression status. Changes in gene expression status include abnormalities in the structure or expression of the transcription product. In other words, in addition to direct detection of genes, RNA can also be selected as the material using reverse transcription PCR ~ Real-time fluorescent quantitative PCR~Northern-blot~ Gene chip and other technologies to detect whether gene expression is abnormal; You can also choose protein as the material and use Western-blot~ Protein histochemical staining~ ELISA and protein chip technology to detect whether gene expression is abnormal. Generally, the deletion of large fragments of DNA often involves multiple genes and leads to a mixture of multiple clinical phenotypes. In addition to cytogenetic or chromosomal analysis, the deletion of larger fragments is usually detected by Southern blot analysis or PCR.

Environmental genome research results are an important basis for molecular diagnosis

Many human diseases are related to heredity ~ nutritional status ~ age ~ various environments and individual development periods. For most chronic diseases, only by clarifying their genetic basis and environmental factors can it be possible to have a thorough understanding of their etiology . People are increasingly aware that genetic background is an important factor that significantly affects disease susceptibility, especially environmentally induced chronic diseases such as tumors ~ asthma ~ diabetes ~ cardiovascular diseases and neurodegenerative diseases are closely related to genetic factors. Therefore, it is a great challenge for modern molecular medicine to fully understand the differences in the susceptibility and genetic background of different individual environmental related diseases from the structure and function. The EGP environmental genome project was launched in 1998. It is the world’s first large-scale human functional genomics system study with genetic polymorphism as an entry point. It can be said to be the second-generation human genome project. According to the arrangement of EGP, human gene resequencing ~ functional variation analysis and the three stages of animal model construction all rely on high-throughput molecular detection methods like molecular diagnosis, and the results of EGP are bound to become an important basis for molecular diagnosis. Therefore, the relationship and cooperation between EGP and molecular diagnostics must be strengthened. Molecular diagnostics must absorb the research results of EGP at any time.

Give full play to the irreplaceable role of molecular diagnosis in overcoming drug resistance treatment and research

Resistance (resistanceD, also known as drug resistance, is the relative resistance of microorganisms to antimicrobial drugs. Drug resistance is divided into intrinsic drug resistance ~ acquired drug resistance ~ multi-drug resistance and cross drug resistance. Scientists have found that bacteria are resistant to Antimicrobial resistance may be spontaneous or it may be a mutation that occurs in bacterial genes through mutations. Such changes allow bacteria to acquire the ability to resist antibacterial drugs and weaken or even inactivate antibacterial drugs. And more importantly, drug resistance. Bacteria can propagate drug-resistant genes not only vertically to their offspring, but also horizontally among different microbial species, which makes many kinds of bacteria develop multi-resistance to different types of antibacterial drugs, which brings many difficulties to clinical treatment. Although there are many factors that affect bacterial resistance, avoiding bacterial resistance is a very complicated problem, but whether it is in the study of microbial resistance mechanisms or in the study of maintaining the usefulness of antibacterial drugs and developing new antibacterial drugs, overcoming microbial resistance The clinical treatment of drug properties requires the use of molecular diagnostic technology to make timely detections. Similarly, although the mechanism of tumor drug resistance is very complicated, the nature of it has not yet been understood. However, it is generally accepted that the failure of chemotherapy is mainly related to the cancer cells’ treatment of chemotherapy drugs. Has or developed drug resistance or multipledrug resistance (multidrug resistance MDR). A variety of molecules or genes related to MDR have been discovered. There is no doubt that a breakthrough in the research of tumor drug resistance is necessary Give full play to the irreplaceable role of molecular diagnostics.

Play the role of molecular diagnosis in disease prediction ~ prevention and individualized treatment

Due to individual differences in genes, each person’s response to a certain food is different, which will cause people to eat the same food but the consequences may be very different. Nutrigenomics is the science that studies the relationship between nutritional intake and the unique genetic code of humans. It is a new health research field created by experts in the fields of biotechnology, genomics, medicine, and nutrition. Genetic testing is a preventive medicine method. In the health check, genetic testing can be used to find out whether a person carries certain susceptible genotypes. Is it easy to get Alzheimer’s disease ~ cardiovascular disease ~ cancer, etc. Then choose different drugs for personalized medicine . Some hospitals or gene companies have begun to provide customers with disease risk predictions (including polygenic genetic diseases and tumor diseases. Clinical drug suitability prediction or nutritional genetic testing. Modern medicine is focusing on safety in addition to prevention and traditional treatment. Effective and the development of economic treatment of drugs. Pharmacogenomics is the study of the relationship between gene mutations and pharmacodynamics. The study of designing drug treatment plans starting with genes has opened up a new way for clinical personalized medicine. Pharmacogenomics is not the study of diseases The genetic factor is to explore the genetic distribution of drug effects to meet clinical needs. Genetic diversity plays a decisive role in the length of individual differences in clinical symptoms ~ costs and clinical treatment efficacy. Pharmacogenomics requires that the production of drugs takes into account the drug delivery area The frequency of the relevant alleles in the population will also vary from person to person and tend to be personalized. The main strategy of pharmacogenomics research includes selecting candidate genes for drug onset ~ activation ~ excretion and other related processes to study and identify gene sequences Variations. These variations can be studied at the biochemical level to estimate their significance in the effects of drugs, and can also be studied in the population. The relationship between genetic mutations and drug effects can be analyzed by statistical principles. From the above analysis, it can be seen that molecular diagnosis is in disease prediction ~ The role of prevention and individualized treatment will become increasingly important.

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