How to make etiological diagnosis of microorganisms in samples?

Abstract: There are many kinds of pathogenic microorganisms, which mutate rapidly, and the inspection technology for rapid identification of pathogenic microorganisms is also developing continuously. At present, the widely used detection methods of pathogenic microorganisms mainly include direct smear microscopy, isolation and culture, biochemical reaction, serological reaction, nucleic acid molecular hybridization, gene chip, polymerase chain reaction and so on. This paper summarizes the progress of these detection technologies. Microorganisms that are pathogenic to humans and animals are called pathogenic microorganisms, also known as pathogens, including viruses, bacteria, rickettsia, mycoplasma, chlamydia, spirochetes, fungi, actinomycetes, prions and so on. These pathogenic microorganisms can cause infections, allergies, tumors, dementia and other diseases, and are also one of the main factors that endanger food safety. In recent years, SARS, highly pathogenic avian influenza, West Nile virus infection and other diseases are highly contagious and often cause worldwide pandemics, so the detection of pathogens must be rapid and accurate. Conventional pathogen detection methods have complex operation, long detection period and high technical requirements for operators. With the continuous development of medical microbiology research technology, pathogen diagnosis is no longer limited to pathogen level, and detection methods at molecular level and gene level are constantly emerging and applied in clinic and laboratory. Detection methods such as nucleic acid molecular hybridization technology, PCR technology and gene chip technology are highly automated, fast, time-saving, pollution-free and accurate, and can identify pathogenic microorganisms accurately and sensitively. 1 traditional detection methods of pathogenic microorganisms The traditional laboratory tests of pathogenic microorganisms mainly focus on dyeing, culture and biochemical identification. Direct smear staining and microscopic examination of specimens and inoculation on culture medium for isolation and culture are common methods for etiological diagnosis of bacterial or fungal infectious diseases. 1. 1 Direct smear microscopy showed that the pathogenic microorganisms were tiny, mostly colorless and translucent, and their size, shape and arrangement could be observed under the microscope after staining. Direct smear staining microscopy is simple and rapid, and it is still applicable to those special forms of pathogenic microbial infections, such as gonococcal infection, mycobacterium tuberculosis infection and spirochete infection. Direct smear microscopy is still a very important means to detect pathogenic microorganisms in grass-roots laboratories without special instruments and equipment. 1.2 separation culture and biochemical reaction separation culture are mainly used to isolate a certain kind of bacteria when there are many kinds of bacteria in clinical samples (such as blood, sputum, feces, etc.). ) or culture. It takes some time for bacteria to grow and reproduce, and the detection period is long, so it is impossible to process batch samples at the same time. In order to solve this problem, various automatic culture identification systems are constantly produced, and the traditional identification methods are gradually improved, which greatly speeds up the inspection. For example, the Microscan WalLCAway automatic microbial analyzer can do bacterial identification and drug sensitivity test at the same time, and detect more than 500 strains. Hypertrophic bacteria such as Streptococcus pneumoniae, Neisseria gonorrhoeae and Haemophilus influenzae. It requires high nutrition and low positive rate of conventional culture. Yong Gang and others added different proportions of glucose, corn starch, growth factors, yeast powder, amino acids and other special inoculants to chocolate culture medium to make a new gonococcal culture medium, which greatly improved the isolation and culture rate of gonococci. Su Shengtong and others added Chinese traditional medicine jujube and adzuki bean to nutrient agar, and cultivated bacteria such as Streptococcus A, Streptococcus B and Streptococcus Pneumoniae, and the growth index was significantly higher than that of blood plate. 1.3 Tissue cell culture Living tissue cell culture is suitable for treating pathogens living in living tissue cells, including viruses, rickettsia, chlamydia, etc. The sensitive tissue cells of different pathogens are different. Take out living cells from animal tissues sensitive to pathogens for primary culture in vitro or subculture of cell lines sensitive to pathogens, and then inoculate pathogens into corresponding tissue cells, where pathogens can multiply and grow, resulting in specific cytopathic effects. Pathogens can also be directly inoculated into sensitive animals, causing specific lesions in corresponding tissues and organs. Pathogens can usually be identified based on these specific lesions. 2 Serological and immunological detection Serological detection is a technology that uses known antibodies or antigens to detect antigens or antibodies of pathogens, thus quickly identifying pathogens and simplifying the identification steps. Commonly used methods include serum agglutination test, latex agglutination test, fluorescent antibody detection technology, synergistic agglutination test and enzyme-linked immunosorbent assay. The application of enzyme-linked immunosorbent assay greatly improves the sensitivity and specificity of serological detection, which can not only detect pathogen antigens in samples, but also detect antibody components of the body. The infection rate of Helicobacter pylori in China population is as high as 50% ~ 80%. Enzyme-linked immunosorbent assay (ELISA) was used to detect anti-HP antibody in saliva to diagnose HP infection, and the result was satisfactory. The infection rate of hepatitis B virus (HBV) is extremely high in China, and the role of ELISA in early serological diagnosis of hepatitis B patients is the most obvious. Clinically, pathogenic bacteria are often mixed with non-pathogenic bacteria, and how to isolate the target bacteria from these bacteria is the key. Immunomagnetic bead separation technology (IMBS) is a new technology developed in the field of microbial detection in recent years. Its basic principle is that monoclonal antibody or polyclonal antibody or secondary antibody of specific pathogen is coupled to magnetic bead microspheres, and magnetic bead-target pathogen complex or magnetic bead-target pathogen complex is formed through antigen-antibody reaction, and the target pathogen is separated under the action of external magnetic field. At present, immunomagnetic beads have been developed for various pathogens, such as Escherichia coli, Listeria, Candida albicans and Legionella, which are widely used in scientific research and laboratories at all levels. Candida albicans isolated by IMBS can be directly detected under the microscope, and the detection time is shortened to 4 hours. IM-BS can also be combined with other detection techniques to detect pathogenic bacteria, and the target bacteria separated by immunomagnetic beads are isolated and cultured, so that the minimum detection limit of E.coli 0 157 is increased from 200 cfu g to 2 cfu g ~；; IMBS combined with polymerase chain reaction (imbs-PCR) can quickly detect bacteria under special culture conditions, such as demanding bacteria and anaerobic bacteria. Detection of Clostridium perfringens, a toxin-producing bacterium in meat by IMBS—PCR