With the completion of the Human Genome Project in 2003, gene sequencing technology continues to evolve, and next generation sequencing (NGS) technology has gradually grown into mainstream sequencing technology. Its continuous innovation in sequencing principles and continuous breakthroughs in technology have made it high. The application of flux gene sequence reading tends to individualized needs, and is expected to lead and replace the commonly used molecular diagnostic techniques.
First, NGS
NGS is also known as high throughput antibody production sequencing, including second-generation massively parallel sequencing and three-generation single-molecule sequencing. The second-generation sequencing includes Illumina's Solexa sequencing platform, Roche's 454 sequencing platform, and ABI's oligonucleotide-linked detection platform (SOLiD). Different platforms have different sequencing methods, but the principles have commonalities, all of which are genomes. The DNA is randomly spliced into small fragments of DNA, and the sequencing template is obtained for sequencing and data analysis by constructing a library, PCR amplification and other technical processes. Compared with Sanger sequencing, the flux is high, the speed is fast, and the cost is low, but the read length is generally Short shortcomings.
The third-generation sequencing is based on the single-molecule real-time technology of American Pacific Biotechnology and the nanopore single-molecule sequencing technology of Oxford Nanopore, UK. It is characterized by no need for PCR amplification, distinguishing base signal differences by different means, and directly reading sequence information. Long reading and high throughput monoclonal antibody generation can directly sequence RNA and methylated DNA sequences. At present, there are shortcomings such as high error rate, high cost and insufficient bioinformatics analysis software.
Second, the current clinical application of NGS in infectious diseases
In recent years, there have been new changes in the spectrum of infectious diseases worldwide. On the one hand, the number of difficult infections is increasing. Traditional diagnostic methods cannot keep up with the rhythm of the continuous evolution and mutation of microorganisms. On the other hand, the spread of infectious diseases is obviously accelerated. These are all infections. The diagnosis and treatment of sexual diseases poses serious challenges. The rapid detection and diagnosis of clinical specimens of infectious pathogens has become an increasingly urgent need in the clinic. NGS technology plays an increasingly important role in infectious diseases because of its high speed, high accuracy and low cost.
1. Application in the detection of bacterial infections:
Clinically, the culture method is a standard for diagnosing bacterial infections. It takes a long time, has high technical requirements, and some bacteria grow harsh conditions, which increases the difficulty of successful culture. NGS technology is applied to the detection of bacterial infections. It can not only determine whether there is bacterial infection, but also directly classify the strains, obtain molecular serotypes, drug resistance gene profiles and other information, and analyze the evolution history, infection source and infection path of the strains. Helps with disease prevention, diagnosis and vaccine development.
In May 2011, Germany was the center of Europe, and a serious hemorrhagic intestinal infection broke out in Europe. Scientists carried out genome-wide sequencing of the pathogen strain and found that the causative culprit was the lethal Escherichia coli O104:H4, through the sequence Analyze the path and pathogenic mechanism of infection in human body, trace the source of infection path, and provide important information for the diagnosis and treatment of the disease. In June of the same year, similar infections occurred in France. It is generally believed that the German strain may be infected. The source, and the two methods of pathogens were completely consistent by traditional methods and molecular diagnostic methods, but the NGS sequence analysis found that the genetic diversity of Escherichia coli strains in Germany is much lower than that of French strains, thus confirming its evolution as a French pathogen. Support, clear source of infection. In 2013, similar cases were reproduced in Europe. After sequencing, the strains were found to be different from O104:H4. However, they have a variety of common pathogenic gene regions, and there is also the possibility of causing serious outbreaks. The sequencing results of related strains are helpful. To establish an effective disease warning.
In 2011, the National Institutes of Health in the United States had a carbapenem-resistant Klebsiella pneumoniae infection. Although early detection of infection prevention measures, 18 patients were infected, 11 infected patients died, comprehensive gene sequencing and epidemiology The analysis traces the source of infection and the route of infection, and promotes the control of nosocomial infection and transmission.
Due to the widespread use of genetic mutations and antibiotics, more and more bacteria have developed resistant strains. Among them, the resistant strains of Mycobacterium tuberculosis are difficult to culture and take a long time, and clinical testing is greatly limited. However, as a single-gene bacteria with low sequence diversity, gene sequencing technology has great advantages in detecting the virulence and drug resistance of its strains, and can quickly detect pathogen infection and its drug resistance, with the continuous maturity of technology. And the decline in cost, is expected to replace the existing culture and molecular diagnostic methods, can also analyze the molecular evolution rate and mode of the infection process through sequencing data, which has far-reaching significance for treatment and vaccine development.
2. Application in virus infection detection:
The diagnosis of viral infection is mainly based on antigen-antibody detection. Molecular diagnostic methods such as PCR detection and DNA probe are also widely used. NGS technology is mainly applied to genotype analysis, drug-resistant mutation detection and virus evolution in the host, as well as outbreaks of infected viruses. For rapid detection to assist with prevention, diagnostic treatment and vaccine research.
Hepatitis B virus is the main cause of chronic liver disease such as hepatitis, cirrhosis and liver cancer. The nucleoside antiviral drugs commonly used in the treatment of viral infections have the advantages of less toxic and side effects and good effects, but the possibility of virus resistance mutations in long-term treatment is resistant. The drug can cause serious cases such as viral rebound and hepatitis attack, even liver decompensation and acute liver failure. The drug resistance gene locus can be detected by NGS, and the treatment plan can be adjusted according to drug resistance in time to achieve better curative effect.
AIDS is a disease that seriously threatens the public health of our country. Its pathogen, HIV, is a highly mutated virus. The current guidelines recommend that the viral load declines or the treatment failure needs to be changed before and during antiviral therapy. The patients in the program were tested for drug resistance, and the results provided an important reference for the development and adjustment of the treatment plan. NGS can detect local HIV genotype resistance mutations, clarify the distribution of drug resistance genotypes and their subtypes, provide guidance for the selection and replacement of clinical therapeutic drugs, and accurately understand the evolution and mutation of HIV in the host. Possibly, it provides a new idea for the design of antiviral drugs and the development of vaccines.
In 2014, West Africa broke out the largest Ebola virus infection in the record, with a long outbreak, high infections and deaths, and caused great panic worldwide. Through the combination of NGS technology and data analysis, the researchers identified their genetic diversity, vulnerability and epidemiological characteristics, tracked the source of infection and the path of infection, and predicted the process of viral infection of the human body and the body's immune response. The evolution of the adaptive mutation in the host reduces the chance of being recognized by immune cells, and has greatly promoted the prevention, control, diagnosis, treatment and vaccine development of the epidemic.
3. Role in the detection of fungal and other pathogen infections:
With the increase in the use of antibiotics, hormones and immunosuppressive drugs, the number of patients with malignant tumors and organ transplants has increased, and the incidence of invasive fungal infections has gradually increased. The mortality rate is over 50%. Rapid and accurate diagnosis is the key to treatment. . Traditional culture methods have become increasingly unable to meet clinical needs. In recent years, with the rapid development of NGS technology as a technical support, the genome-wide data of a series of important pathogenic fungi have been published one after another, making humans aware of these pathogenic fungi. To a whole new level. Through gene sequencing detection, the establishment of local fungal genomics data and comparative analysis can not only carry out rapid and accurate diagnosis of fungal infections, but also discover functional genes unique to different fungi, study its pathogenic mechanism and drug resistance site mutations, and discover new ones. The drug acts on the target site, thereby developing an antifungal drug with a broad spectrum, low adverse reactions, and no cross-resistance.
In the clinical detection of other pathogen infections other than bacteria, viruses and fungi, NGS currently plays a complementary role. When clinical and laboratory are unable to identify pathogens, high-throughput sequencing technology and bioinformatics analysis can be used to confirm the diagnosis. Infect pathogens and establish a local database for rapid diagnosis and further clinical research. In 2009, China completed the whole genome sequencing and functional analysis of the first multi-cellular human parasite, analyzed the composition of its functional genes, and explored the process of its co-evolution with the host, which effectively promoted the diagnosis, treatment and prevention of schistosomiasis; In addition, domestic and foreign scholars also through the genome-wide sequencing of Plasmodium, mapping its genetic map, studying the genetic differences and mutations of different Plasmodium, providing ideas for the development of new drugs and vaccines.
4. The clinical significance of metagenomic detection:
The human body has a large number of microorganisms that play an indispensable role in metabolic activities, researching microorganisms in the human body, exploring its interaction with the human body, and the relationship between microorganisms and diseases, exploring new ways of disease prevention and control, and promoting Human health is of great significance. Most microorganisms cannot be studied by traditional culture and identification. Metagenomics is a discipline that studies the genome of microbial populations. The technology does not rely on the cultivation of microbial communities in the sample, but directly performs nucleic acid extraction and determines all the samples in the sample. The nucleic acid sequence of the microorganism is analyzed and functionally identified. It not only can understand the microbial community's own quantity and composition, but also the microbial community itself and its evolutionary relationship with the host, and study the microbial community structure and function under different states. The effects of analyzing the structural and functional characteristics of normal human flora and disease states.
Intestinal microbial metagenomics is closely related to human metabolism and immunity. It has become a hot research topic in recent years. The relationship between intestinal flora and metabolic diseases such as diabetes and obesity has been continuously studied. The interaction between drugs and intestinal microflora is also being explored.
Third, NGS technology is currently used in clinical testing problems
There are still many practical problems in the routine application of NGS technology in clinical tests. The complexity of clinical specimens may result in too little pathogen information and data loss. Pathogen data may be mixed in the normal flora and it is difficult to distinguish. There are no different specimens yet. Uniform provisions for pre-sequencing processing and parameter setting; high-throughput sequencing will generate large amounts of data, requiring huge data analysis. If there is a lack of local databases, the cost and time of computing work is difficult to control; for disease, sequencing of genomic data The relationship with disease is still unclear. Access to genomic information alone is not sufficient to clarify the cause of the disease and the process of disease development. Accurate analysis and data mining are needed to decipher the causes of infectious diseases and achieve precise treatment and intervention. Further exploration is needed to ensure the accuracy of the application in clinical practice.
With the continuous updating of sequencing technology, the cost of antibody sequence database will continue to decrease. Once a complete genetic database is established, NGS technology will make greater achievements in the identification of pathogenic microorganisms, reduce the occurrence and spread of infectious diseases, and become a routine clinical test. Technology to promote medical development.
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