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Molecular diagnosis is a method and process that uses DNA, RNA, or protein molecules as diagnostic materials to specifically diagnose the state of the human body or diseases by examining the presence, defects, or abnormal expression of endogenous genes or exogenous (pathogen) genes. With the implementation of the Human Genome Project and the post-genome (proteomics) project, molecular diagnostics have been further improved and matured. The combination of molecular biology and modern medicine has expanded laboratory diagnostics far beyond merely assisting clinical diagnoses, playing an increasingly significant role in disease prevention, prognosis assessment, monitoring treatment efficacy, evaluating health status, and predicting diseases. It is not an exaggeration to say that molecular diagnosis is a groundbreaking testing method that will significantly impact the development of medicine. Today, molecular diagnosis has become an important indicator of the overall healthcare level of a country or region. Therefore, it is crucial to carefully consider, analyze, and summarize the basic strategies of molecular diagnosis. Understanding the strategies of molecular diagnosis is beneficial for consolidating the current research results in molecular diagnostics, further enhancing its role in clinical medicine, and clarifying the direction of development in molecular diagnostics.
1. Molecular diagnosis must adhere to the basic principles of experimental diagnostics.
Medical diagnostics is a science that studies the basic principles and methods of diagnosing diseases. As a new branch of laboratory diagnostics, molecular diagnosis should also consistently follow the fundamental principles of experimental diagnostics. This means analyzing the sent materials (including DNA, RNA, and proteins) from the human body in the laboratory to identify, confirm, and interpret the causes, pathogens, and conditions of diseases, providing objective evidence for clinical doctors to accurately validate clinical impressions, analyze conditions, and explore the occurrence and development of diseases. Although molecular diagnosis is also applied in areas such as resistance testing, efficacy monitoring, health prevention, health checks, disease prediction, and personalized treatment, it still needs to adhere to the aforementioned basic principles of experimental diagnostics. The development of molecular diagnostic technology holds enormous business opportunities. Therefore, in order to safely, effectively, and legally carry out research and applications in molecular diagnosis, it is essential to actively implement patent laws.
2. Different molecular diagnostic strategies and methods should be adopted for different types of diseases and their possible causes.
Infectious diseases are caused by the invasion of pathogens such as bacteria, viruses, mycoplasma, chlamydia, and parasites. Previously, microbiological, immunological, and hematological methods were mainly used to detect these pathogens, but these methods are limited by sensitivity and specificity, making early diagnosis difficult. With the elucidation of the genetic structures of various pathogens, it has become possible to use molecular diagnostic technology to detect infectious pathogens (RNA or DNA) early, rapidly, sensitively, and specifically. By directly determining these pathogen genes using molecular techniques, not only can microbial infections be accurately diagnosed, but carriers or potential infections can also be identified, and typing and resistance monitoring of infectious pathogens can be performed. Therefore, the molecular diagnostic targets for infectious diseases mainly include the exogenous biological DNA or RNA of infectious viruses, bacteria, parasites, mycoplasma, and chlamydia present in the human body. From a theoretical standpoint, such tests can generally be conducted quickly, conveniently, and accurately using PCR or RT-PCR methods based on the known sequences of exogenous DNA or RNA. Thus, the application of genetic diagnosis for these diseases is very strong and can be widely used in clinical settings under strict quality control. The diagnostic strategies can be divided into two types: one is a general detection strategy, which only needs to indicate whether there is an infection by a particular pathogen; the other is a comprehensive detection strategy, which not only diagnoses the pathogen but also conducts typing (including subtypes) and resistance testing. All hereditary diseases are related to mutations in one or more genes. There are two strategies for molecular diagnosis of hereditary diseases: 1. Direct diagnostic strategy (DNA sequence analysis of disease gene mutations), which directly reveals various genetic defects that lead to hereditary diseases; 2. Indirect diagnostic strategy (other diagnostic methods besides DNA sequence analysis of disease gene mutations), which identifies the allelic types and haplotypes of chromosomes related to genetic defects in probands and then looks for whether other family members have the same allelic types and haplotypes. Furthermore, conducting prenatal genetic diagnosis based on hereditary disease risk assessment is a good way to prevent genetic diseases and control population quality. The causes of malignant tumors arise from both endogenous and exogenous factors. Endogenous factors refer to internal structural and functional changes in the body, such as genetic, immune deficiencies, and metabolic abnormalities; exogenous factors refer to various carcinogenic factors in the natural environment. The occurrence and development of tumors result from the synergistic effects of multiple factors, with abnormalities at the genetic level being a key reason. In this sense, tumors are genetic diseases. Currently, molecular diagnosis of tumors can adopt three strategies: 1. Detecting tumor-related genes; 2. Detecting the genes of tumor-related viruses; 3. Detecting tumor markers. Essentially, providing information on tumor-related genes can be classified as direct diagnosis, while detecting tumor-related viral genes or tumor markers mainly falls under indirect diagnosis.
3. Conduct different levels of molecular diagnosis based on the purpose of genetic testing.
The basic strategies of molecular diagnosis are closely linked to the causes of diseases. The causes of human diseases include two main categories: internal causes and external causes. Internal causes mainly refer to genetic factors, such as changes in gene structure and gene expression status. Changes in gene structure include point mutations, insertions, deletions, rearrangements, translocations, and polymorphic variations in gene structure; external causes refer to external environmental factors, such as lifestyle, work environment, mental state, and the invasion of various infectious pathogens. Based on the purpose of genetic diagnosis, five basic methods can be chosen.
3.1 Chromosome Analysis
For large segment DNA detection, besides using traditional cytogenetic techniques, more advanced chromosome analysis techniques such as fluorescence in situ hybridization (FISH) can be used. Probes such as cosmid, bacterial artificial chromosomes (BAC), or yeast artificial chromosomes (YAC) can achieve very stable hybridization effects. Comparative genomic hybridization (CGH) technology can partially compensate for the limitations of the above techniques, overcoming the restriction that the information provided is limited to the areas covered by the probes.
3.2 DNA Content Measurement
DNA content measurement is also part of DNA analysis. Cells with abnormal numbers of nuclear DNA often carry multiple genes (including those regulating growth and apoptosis). This can be detected using flow cytometry. Changes in DNA content (DNA index) relative to normal cells may indicate the presence of an increase or loss of a particular chromosome (aneuploidy) or an extra set of chromosomes (polyploidy).
Additionally, the DNA content spectrum of tissue cells can reflect the ratio of S-phase to G2-phase cells. In certain tumors (such as breast cancer), a high ratio may indicate a poorer prognosis.
3.3 Detection of Gene Mutations
If a specific mutation in a pathogenic gene has a direct causal relationship with the disease, using this gene mutation as a diagnostic basis is ideal. For pathogenic genes that are fully or partially understood, direct detection and analysis of mutations is the most powerful and accurate method of molecular diagnosis. Since one gene can have different mutation types, it is necessary to develop a reasonable and convenient strategy for the individual being tested. Generally, large segment deletions of DNA often involve multiple genes, leading to various mixed clinical phenotypes. Therefore, besides chromosome analysis, Southern blotting or PCR detection is typically used for larger segment deletions. Detection of small segment insertions and deletions, as well as point mutations, is mainly due to the fact that pathogenic genes can mutate at any site, resulting in different mutation types for the same disease. With the advent of PCR technology, designing primers based on known gene sequences to directly detect the object of observation has become very convenient. Furthermore, using PCR combined with single-strand conformation polymorphism analysis (PCR-SSCP) can sensitively detect single point mutations. Although gene mutation detection has direct advantages, many pathogenic genes have not yet been cloned, and while some pathogenic genes are known to be located on chromosomes, their exact gene structures and molecular mechanisms are still poorly understood. Therefore, many diseases cannot adopt the mutation detection route.
3.4 Gene Linkage Analysis
Due to the linkage relationship between closely located genes on the same chromosome, linkage analysis should be conducted for pathogenic genes that have not yet been identified but are at least localized to specific chromosomal regions. By identifying the presence or absence of genes linked to the pathogenic gene, it can be determined whether the subject carries the pathogenic gene. Molecular polymorphic markers can be used to track the separation of mutation alleles within a family. This type of analysis not only requires collecting DNA samples from probands but also from two to three generations of affected and unaffected family members. To minimize errors caused by recombination, the polymorphic markers must be sufficiently close to (or located within) the pathogenic gene. Classical linkage analysis mainly uses restriction fragment length polymorphism (RFLP) as genetic markers for family analysis. In recent years, STR (short tandem repeat) has rapidly developed as a DNA polymorphic marker for linkage analysis, gradually replacing RFLP. Studies have shown that except in a few cases, the error rate due to chromosomal recombination is less than 1%. Therefore, DNA marker linkage analysis is generally reliable. To ensure the accuracy of genetic diagnosis through linkage analysis, it is necessary to search for 2-3 genetic markers on both sides of the detected gene or locus, thus allowing haplotype analysis to prevent biases caused by recombination.
3.5 Detection of Gene Expression Status
Molecular diagnosis can detect not only structural abnormalities of genes but also gene expression status. Changes in gene expression status include abnormalities in the structure or expression levels of transcription products. In other words, besides directly detecting genes, RNA can be selected as the material to utilize techniques such as reverse transcription PCR, real-time quantitative PCR, Northern blot, and gene chips to detect whether gene expression is abnormal. Protein can also be selected as the material to use techniques such as Western blot, protein immunohistochemistry, ELISA, and protein chips to detect whether gene expression is abnormal. Generally, large segment deletions of DNA often involve multiple genes, leading to various mixed clinical phenotypes. In addition to using cytogenetic or chromosome analysis, larger segment deletions are typically analyzed using Southern blotting or PCR detection.
4. Environmental genomics research results are an important basis for molecular diagnosis.
Many human diseases are related to genetics, nutritional status, age, various environmental factors, and individual developmental stages. For most chronic diseases, only by clarifying the contributions of genetic and environmental factors can a thorough understanding of their etiology be achieved. There is an increasing recognition that genetic background is a significant factor influencing disease susceptibility, especially for chronic diseases induced by the environment, such as tumors, asthma, diabetes, cardiovascular diseases, and neurodegenerative diseases, which are closely related to genetic factors. Therefore, fully understanding the differences in susceptibility to environment-related diseases among different individuals, in terms of structure and function, and seeking related susceptibility genes is a significant challenge faced by modern molecular medicine. The Environmental Genome Project (EGP) was initiated in 1998, representing the world’s first large-scale human functional genomics study focusing on genetic polymorphisms, and can be considered the second generation of the Human Genome Project. According to the EGP’s arrangements, the three phases of human gene resequencing, functional variation analysis, and animal model construction, like molecular diagnosis, rely on high-throughput molecular detection methods, and the results of the EGP will inevitably become an important foundation for molecular diagnosis. Therefore, it is essential to strengthen the connection and mutual cooperation between EGP and molecular diagnosis, and molecular diagnosis should continuously absorb the research results of EGP.
5. Fully leverage the irreplaceable role of molecular diagnosis in overcoming resistance treatment and research.
Resistance, also known as drug resistance, is the relative resistance of microorganisms to antimicrobial agents. Resistance can be classified into intrinsic resistance, acquired resistance, multidrug resistance, and cross-resistance. Scientists have found that bacteria can develop resistance to antimicrobial agents either spontaneously or through mutations. These mutations occur in bacterial genes and allow bacteria to gain the ability to resist antimicrobial agents, diminishing or even inactivating the activity of these drugs. More importantly, resistant bacteria can reproduce and transmit resistance genes not only vertically to their offspring but also horizontally between different species of microorganisms, leading to multiple bacteria developing multidrug resistance to different classes of antimicrobial agents, thus posing significant challenges for clinical treatment. Although many factors influence bacterial resistance, avoiding bacterial resistance is a complex issue. Nonetheless, whether in the study of microbial resistance mechanisms or in maintaining the efficacy of antimicrobial agents and developing new antimicrobial agents, timely detection through molecular diagnostic techniques is essential in overcoming microbial resistance in clinical treatment. Similarly, although the mechanisms of tumor resistance are complex and not yet fully understood, it is widely recognized that the failure of chemotherapy is mainly related to cancer cells developing or acquiring resistance or multidrug resistance (MDR). Many molecules or genes related to MDR have been identified. Undoubtedly, to achieve breakthroughs in the study of tumor resistance, it is crucial to fully utilize the irreplaceable role of molecular diagnosis.
6. Leverage molecular diagnosis in disease prediction, prevention, and personalized treatment.
Due to genetic differences, individuals react differently to certain foods, which can result in significant variations in outcomes even when consuming the same food. Nutritional genomics is a field of science that studies the relationship between nutrient intake and the unique genetic codes of humans, created through the collaborative research of experts in biotechnology, genomics, medicine, and nutrition. Genetic testing is a preventive medical measure that can identify whether an individual carries certain susceptible genotypes, such as those prone to Alzheimer’s disease, cardiovascular diseases, or cancer, thus allowing for the selection of different drugs for personalized medical treatment. Some hospitals or genetic companies have started offering disease risk predictions (including multi-gene hereditary diseases and tumor diseases) in health checks, along with clinical drug suitability predictions or nutritional gene testing. Modern medicine is evolving towards safe, effective, and economically viable drug treatments, in addition to focusing on prevention and traditional therapies. Pharmacogenomics, which studies the relationship between gene mutations and drug efficacy, opens new avenues for designing drug treatment plans based on genetics, focusing on the genetic distribution of drug effects to meet clinical needs. Genetic diversity plays a decisive role in individual differences, clinical symptoms, treatment costs, and effectiveness. Pharmacogenomics requires drug production to consider the frequencies of relevant alleles in the local population, leading to personalized medical prescriptions. The main strategies in pharmacogenomics research include selecting candidate genes related to drug activation, effect, and elimination processes, and identifying gene sequence variations. These variations can be studied at the biochemical level to estimate their significance in drug action or analyzed in populations using statistical principles to explore the relationship between gene mutations and drug efficacy. From the above analysis, it can be seen that the role of molecular diagnosis in disease prediction, prevention, and personalized treatment will become increasingly important.
7. Strive to develop simple, rapid, accurate, and instrument-free molecular detection methods.
Modern medical testing laboratories, whether independent or part of healthcare units, are entities equipped with many high-tech and complex instruments and trained professionals. This setup reflects the complexity and precision of modern medical testing operations and underscores the significant role of modern medical testing in medical practice. However, the configuration of advanced instruments and specialized personnel also imposes a heavy economic burden on patients. A report from the World Health Organization’s Collaborating Center for Bioethics indicated that 90% of medical research funding is allocated to address health issues faced by only 10% of the world’s population. In recent years, the results of biotechnology research have increased significantly and have been widely applied in medical practice. However, most of this research prioritizes developed countries, with only a few studies proving effective in improving healthcare conditions in developing countries. According to the voting results of ten important biotechnologies for developing countries decided by an expert committee, improving molecular detection technologies and developing low-cost, simple infectious disease diagnostic reagents received the highest votes. The World Health Organization’s special program for tropical disease research and training aims to promote and assist in developing, evaluating, and promoting diagnostic reagents suitable for primary healthcare units in developing countries. The ideal diagnostic reagents set by the World Health Organization for primary healthcare units in developing countries are affordable, sensitive, specific, user-friendly, rapid, robust, equipment-free, and deliverable. The first letters of these seven words spell out ASSURED, which implies certainty. With the completion of the Human Genome Project and the implementation of the international HapMap project, a solid foundation has been laid for researching the relationship between specific diseases and genetic variations. Currently, there are many misconceptions in biomedical research in our country, and there is an urgent need to strengthen large-scale prospective cohort studies to promote the advent of a new era of prediction, prevention, and personalized medicine. This is a great opportunity for molecular diagnostic technology to shine. In response to the threat of biological terrorism, there is an urgent need to develop low-cost, simple, and rapid gene diagnostic reagents with Chinese characteristics. Based on the above spirit, the research and development of low-cost, simple, rapid, accurate, and instrument-free molecular detection methods is imminent.
8. Pay close attention to medical ethics and biosafety issues in molecular diagnosis.
Due to the rapid development of molecular diagnostic technology and the advancement of social awareness, the use of human tissues for diagnostic and analytical research is becoming increasingly complex. The results of gene diagnosis may lead to the inclusion of a large amount of potentially relevant information about disease susceptibility, pharmacogenomics, nutritional genomics, and other biological data in the expanding biological database. When using archived tissue samples and biological information for analytical research and validation, the privacy and informed consent of patients or providers of tissue samples and biological information must be seriously considered. The use of tissue samples and biological information must be handled under the guidance of relevant medical ethics institutions and research review committees, following established systems or guidelines. Biosafety issues during the collection, handling, and storage of human biological samples for gene diagnosis must also be given high priority and must be managed according to systems or guidelines. Additionally, it is important to note that biosafety issues are not limited to infectious diseases; potential risk factors hidden in biological samples from other diseases should not be overlooked.
9. Strengthen quality control of gene diagnostic technology.
When molecular biology technologies or methods are used for gene diagnosis, they must be placed under the quality control of clinical laboratories. Using quality-certified reagent kits is a common practice in routine clinical laboratory testing. This means that before a testing method is officially used, it must first be evaluated from both theoretical and clinical perspectives, followed by the formulation of specialized technical specifications. However, many molecular testing methods are operated in different laboratories in their own ways before they have undergone evaluation or quality certification. Therefore, quality control of these tests should be given special attention. Another important aspect of quality control is the skill level of operations, which includes the qualification certification of personnel engaged in molecular testing. In summary, quality control should be implemented at every stage of gene diagnosis, from the clinical collection of tested samples to laboratory transport, preparation, testing analysis, and the interpretation and reporting of results. Let us work together to grasp the strategies of molecular diagnosis for the prosperity of molecular diagnostics and contribute to developing more low-cost, simple, rapid, accurate, and instrument-free molecular diagnostic reagents for primary healthcare units in China and all developing countries.
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