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Research progress of rapid diagnosis technology of clinical microbiology test
Keywords: Molecular Biology Source: CHKD Journal Full-text Database Contemporary Medicine,No. 16, 2009.
(Author: Li Suli, No.252 Hospital of the People's Liberation Army)
At present, infectious diseases are still a major hidden danger to human health. With the emergence of new and sudden infectious diseases, infectious diseases that have been controlled have come back, and the types of microorganisms that cause infectious diseases are becoming more and more complicated. The threat of common pathogenic microorganisms has not been eliminated, but a large number of drug-resistant strains have appeared, and the emergence of new pathogenic microorganisms has brought great trouble to clinical diagnosis and treatment. The severe reality puts forward higher requirements for the detection and diagnosis of pathogenic microorganisms. The World Health Organization (WHO) proposed to the clinical microbiology laboratory that it should focus on rapid diagnosis as much as possible. Use all means to transform laboratory data into clinically useful information.
1 application of automatic identification technology
Laboratory examination of clinical microorganisms is mainly based on staining, culture and biochemical identification, especially isolation and culture, which is still the "gold standard" for many pathogens detection. However, it takes some time for bacteria to grow and reproduce, so it is difficult to shorten the detection period. In addition, the culture of many pathogens is affected by nutritional requirements, antibiotic application and pathogen content. The traditional manual method has complicated operation, long detection period and limited sensitivity and specificity. In order to solve this problem, various automatic cultivation and identification systems are constantly produced. With the development and application of computer, many automatic and semi-automatic bacterial identification and drug sensitivity systems have appeared, which are collectively called "expert system for microbial identification". These systems have greatly improved the working efficiency and detection accuracy of clinical laboratories, and the traditional identification methods have been gradually improved, which has accelerated the detection speed to a certain extent.
2 Immunological methods
Immunology technology uses specific antigen-antibody reaction to detect pathogenic microorganisms, which simplifies the identification steps of pathogenic microorganisms and attracts much attention. The data provided by major literature databases show that almost all serological detection methods for pathogens have been established, indicating that this method has become a mature detection technology commonly used in microbiology laboratories.
2. 1 agglutination technology The commonly used agglutination technologies are latex agglutination technology and serum agglutination technology. Used for preliminary diagnosis, typing and identification of microorganisms, such as typing of Vibrio cholerae and Shigella, Escherichia coli O.57: H7, meningococcus, etc. And the identification can be completed in a short time. The diagnosis method is simple, rapid, accurate, specific and has a high positive rate.
2.2 Fluorescent antibody technology Fluorescent antibody technology is based on the high specificity of antigen-antibody reaction, using fluorescein as antigen marker, and examining specific antigen-antibody complexes and their existing parts under fluorescent microscope. The main characteristics of fluorescent antibody technology are strong specificity and high speed. Lv Zhilin and others reported that monoclonal antibodies labeled with cell wall specific fluorescence (CW-DFA) and capsule antigen (CAP-DFA) made by American colleagues can quickly identify Bacillus anthracis.
2.3 Enzyme immunoassay Enzyme-linked immunosorbent assay (ELISA) has been widely used to detect a variety of pathogenic microorganisms, which can detect pathogenic antigens in samples and antibody components in vivo. The possible mycoplasma pneumoniae, influenza virus, parainfluenza virus, respiratory syncytial virus and adenovirus were successfully detected from throat swab samples of patients by monoclonal antibody on nitrocellulose membrane combined with dot enzyme-linked immunosorbent assay. Gehring et al. identified Escherichia coli O.57: H7 by ELISA. Many diseases have been detected by commercial kits.
3 molecular biology technology
With the rapid development of molecular biology technology, people's understanding of microorganisms has gradually shifted from external phenotype to internal gene structure characteristics, and microbial detection has also shifted from biochemical and immune methods to gene level detection. For those microorganisms that are difficult and impossible to cultivate, genetic information can be obtained directly, which brings a new field to microbial detection and provides new opportunities for the rapid development of science.
3. 1 PCR technology has high sensitivity and specificity. In pathogen detection, it is often difficult to accurately detect microorganisms with atypical morphology and biochemical reaction by conventional methods, and even if a large number of dead bacteria appear, PCR can make accurate identification; Without the influence of mixed samples, pathogens can be easily identified from samples containing a large number of normal flora; Used to identify microorganisms that grow slowly or are difficult to culture, such as Mycobacterium, Helicobacter pylori, Mycoplasma, Chlamydia, Spirogyra, etc. At present, the positive detection rate of other methods is very low, and PCR technology is of great significance to the identification of such strains.
However, conventional PCR technology has some problems, such as false positive, primer dimer formation, complex detection operation and many intermediate pollution links, which are prone to false positive or false negative results. In order to overcome these shortcomings, some new PCR techniques are gradually derived and applied in practice, such as nested PCR, reverse transcription PCR, multiplex PCR, universal primer PCR(UP-PCR), PCR single strand conformation polymorphism analysis, random primer DNA polymorphism amplification (RAPD), restriction length polymorphism analysis (RFLP), real-time fluorescence quantitative PCR and so on.
3.2 detection technology based on 16S rRNA and GyaB
3.2. 1 detection 16S rRNA as the target gene 16S rRNA exists in all prokaryotic cells. They are relatively stable and have high copy number, and their sequences contain variable regions and highly conserved regions, so group, genus and species-specific probes can be designed. At present, almost all the 16S rRNA genes of various common bacteria have been sequenced. These characteristics of 16SrRNA coding gene make it an ideal target sequence for bacterial gene classification, and gradually become the "gold standard" for bacterial identification and classification.
3.2.2 Using gyrase B subunit gene target gene to detect GyaB not only has the advantages of 16S rRNA, but also has a higher gene evolution rate than ribosome gene, and it exists in single copy form in almost all bacteria. The research shows that the evolutionary map based on GyaB sequence is consistent with that based on DNA-DNA hybridization. Therefore, the analysis of GyaB is especially suitable for the differentiation and identification of strains. Fukushima et al. designed a gene chip with GyaB gene as the target gene to detect mycobacterium. The experimental results show that the chip can identify mycobacteria at the species level, and can distinguish strains with close genetic relationship, which has important reference value for clinical treatment. It shows that analyzing GyaB gene sequence is a rapid and effective method to identify bacteria at the species level.
3.3 Multi-locus sequence typing Multi-locus sequence typing (MLST) is a molecular biological analysis method that has developed rapidly in recent years. It has high resolution and is suitable for molecular epidemiology and molecular evolution. More and more people in the world use MLST as a common tool to compare strains, establish a more accurate analysis system and method, and study the epidemiological analysis of diseases and population structure caused by different antibiotic-resistant strains, related special genotypes and new mutations.
3.4 Loop-mediated isothermal amplification (LAMP) Loop-mediated isothermal amplification (LAMP) is a sensitive, specific, convenient and rapid nucleic acid amplification technology developed in recent years. Compared with the traditional method, isothermal amplification does not need thermal cycle, and because a large number of white magnesium pyrophosphate precipitates are produced in the reaction, the amplification products can be judged by naked eye observation or turbidimeter without electrophoresis. Therefore, LAMP is a DNA amplification method with strong specificity and high sensitivity, and the results can be judged by naked eyes without thermal cycler. In this method, four specific primers were designed for six regions of the target gene, and the nucleic acid amplification reaction was completed at constant temperature (about 65℃) for about 60min with strand displacement polymerase, and the characteristic trapezoidal bands were amplified. By designing two loop primers, the reaction speed can be increased by 1/2 ~ 1/3. LAMP technology can be used for rapid detection of pathogenic microorganisms in the field, and can be popularized and applied in the field and grassroots in wartime.
4 molecular biosensor
Molecular biosensor is a new technology combining emerging sensor technology with molecular diagnosis technology, and it is a new direction of modern clinical diagnosis development. Because of its accurate detection and simple operation, biosensor has made great progress in many fields in recent years, and has been widely used in the diagnosis of infectious diseases, drug screening, biological warfare agent monitoring and so on, among which DNA biosensor is the most commonly used pathogen detection sensor in clinic. China scientist Chen Jianzhu's latest biosensor can detect the existence of trace SARS virus, smallpox virus and anthrax in just 20 seconds, thus achieving the purpose of early diagnosis.
5 gene chip
Gene chip is a Qualcomm quality index analysis system. Gene chip technology is a brand-new technology. Because it has the advantage of checking tens of thousands of genes at a time, it is regarded as the greatest invention in the study of gene function. It takes many specific oligonucleotide fragments or gene fragments as probes, which are regularly arranged and fixed on solid media such as silicon wafer, glass sheet and nylon membrane to form a biomolecule lattice, so as to achieve the purpose of simultaneously detecting multiple diseases or samples in one test. Based on the characteristics of Qualcomm quantity, miniaturization and parallel analysis, gene chip plays an increasingly important role in the research fields of microbial pathogen detection, species identification, functional gene detection, genotyping, mutation detection and genome monitoring. At present, the genome sequencing of many pathogens such as bacteria and viruses has been completed. Many special genes representing various microorganisms are made into 1 chip. The expression and expression level of pathogen genes in samples can be detected by reverse transcription, so as to judge the pathogen, infection process and host reaction of patients, which greatly improves the detection efficiency.
6 protein fingerprint technology
Protein fingerprint technology is a new technology with the rise of protein omics. In recent years, more and more scholars have realized the necessity of studying microorganisms from protein level. Protein is the executor of bacterial function. There are many kinds of bacteria, and different protein types determine the ever-changing functions and characteristics of bacteria. In 1996, Clayin et al. successfully identified Gram-negative and Gram-positive bacteria by MALDITOF MS mass spectrometry, indicating that different genera and species of bacteria have different protein fingerprints, and the same bacteria has similar protein fingerprints. According to the protein fingerprint of bacteria, bacteria can be quickly identified. Researcher Tang Hong from Institute of Microbiology, China Academy of Sciences, etc. The latest "protein quality fingerprint" technology and its detection method can be used to analyze the changes of protein composition in serum of SARS and non-SARS patients. This method of detecting SARS virus has been clinically confirmed by Beijing Tiantan Hospital. The positive rate is close to 95%, and the specificity is close to 96%. Satisfactory test results can be obtained on the first day of patients' fever. Some experts said that protein fingerprint technology marked the birth of an epoch-making diagnostic model.
With the continuous development of computer technology, the detection of clinical pathogens will develop in two directions: highly automated and simple and rapid detection technology. Through the use of automated instruments, molecular biology technology will play a great role in the diagnosis and identification of pathogenic bacteria and the detection of drug-resistant genes. With the advent of multidisciplinary era, the status quo and traditional concept of clinical pathogen detection will be completely changed, and high efficiency, high quality and rapid unification will be realized.