Review of Immunodiagnostic Techniques for Pathogenic Microorganisms

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. Imbs-PCR can shorten the detection time of Clostridium perfringens in meat to 10 h, and the minimum detection limit can reach 10cfu g~ ~. Some researchers use imbs combined with real-time fluorescence quantitative PCR (imbs-PCR). Leon—Velarde et al. used IMB8 combined with enzyme-linked detection to greatly improve the detection efficiency of Salmonella. 3 Gene detection With the development of science and technology, molecular biological detection technology is changing with each passing day. The identification of pathogenic microorganisms is no longer limited to the general examination of external morphological structure and physiological and biochemical characteristics, but goes deep into the molecular level and nucleic acid level. The nucleic acid sequences of pathogenic microorganisms, that is, gene fragments, are specific and different from other species or genera. Detection of their unique gene fragment sequences can be used to identify pathogenic microorganisms. With the development of science and technology, gene detection technology has gradually replaced other detection technologies and become the mainstream technology for pathogen detection in clinical laboratories and basic laboratories. 3. 1 nucleic acid hybridization technology The process in which a single nucleotide chain with a certain complementary sequence forms a heteroduplex in liquid or solid phase according to the principle of base complementary pairing is called nucleic acid hybridization, and both sides of hybridization are the probe used and the nucleic acid to be tested. In the detection of pathogenic microorganisms, nucleic acid molecular hybridization mainly includes two kinds: membrane blot hybridization and nucleic acid in situ hybridization. Imprinted hybridization on membrane refers to the separation of nucleic acid from microbial cells, purification in vitro and binding to a solid support, and hybridization with labeled nucleic acid probes in liquid phase. In-situ hybridization of nucleic acids means that the labeled nucleic acid probes directly hybridize with nucleic acids in cells or tissue sections. It is also possible to identify pathogens under a fluorescent microscope or a laser scanning focusing microscope with a fluorescent labeled probe, and it can also display the position in three-dimensional space. Compared with other methods, nucleic acid molecular hybridization detection technology has obvious advantages of simplicity, sensitivity, rapidity and specificity. Wong et al. used two different fluorescently labeled oligonucleotide probes to detect Pseudomonas and Acinetobacter from blood samples. The lowest detection limit is 10 cfu mL~ ~, the specificity is 100%, and the detection time is less than 2 h. The oligonucleotide probe is designed for pathogen-specific gene sequences, which can locate pathogens to be detected at different classification levels, such as families, genera, species and subspecies. (Progress in detection technology of pathogenic microorganisms) 3.2 DNA chip, also known as DNA microarray or DNA chip, is a kind of J of biochip and an extension of nucleic acid molecular hybridization technology. Through micromachining technology, tens of thousands or even millions of gene probes, that is, DNA fragments, are regularly arranged into a two-dimensional DNA probe array, fixed on solid supports such as silicon wafers and glass slides, and hybridized with labeled sample molecules for gene detection. Its sequencing principle is the same as that of nucleic acid hybridization, but it solves the shortcomings of traditional nucleic acid hybridization technology, such as complicated operation, low detection efficiency and low automation. The application of gene chip in the diagnosis of pathogenic microbial infection greatly shortens the time required for diagnosis, and can detect whether pathogens are resistant to antibiotics, resistant to those antibiotics and sensitive to those antibiotics. The gene chip designed by Naas can detect various B- lactamase resistance genes in Pseudomonas aeruginosa, Enterobacter and Bordetella. The gene chip designed by Cai Ting et al. detected the resistance rate of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacter cloacae to l7 kinds of antimicrobial agents. The gene chip developed by Batchelor et al. can detect Escherichia coli and Salmonella encoding 47 drug-resistant genes such as spread spectrum 8. Lactamases, sulfonamides, tetracyclines and aminoglycosides. There are also some problems to be solved in gene chip technology, such as improving the specificity of the chip and the sensitivity of detection signals, reducing the manufacturing cost of the chip, and most chips need expensive detection instruments. These problems make gene chip mainly limited to laboratory research and not widely used in the detection and identification of clinical pathogenic microorganisms. (Progress in detection technology of pathogenic microorganisms) 3.3 PCR technology Polymerase chain reaction (PCR) is a technology that uses known oligonucleotide primers to guide and amplify a small number of gene fragments to be detected in vitro. Because PCR can amplify the gene to be detected, it is especially suitable for early diagnosis of pathogen infection, but if the specificity of primers is not strong, it may cause false positive. PCR technology has developed rapidly in recent 20 years, and its reliability has gradually improved from gene amplification to gene cloning and transformation and genetic analysis. Jbara directly detected Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae in 75 samples by PCR and traditional methods. Compared with traditional methods, the specificity and sensitivity of PCR detection were 87.3% and 100%, respectively. In 1988, Chamberian and others put forward the concept of multiplex PCR. Adding more than two pairs of primers to the same PCR reaction system can amplify multiple nucleic acid fragments at the same time, which is suitable for the analysis and identification of a large number of samples. Multiplex PCR has the following advantages: (1) high efficiency, which can simultaneously detect multiple pathogenic microorganisms in the same reaction system or classify different types of the same pathogenic microorganism; (2) Systematic and multiplex PCR are very suitable for detecting group pathogens, such as simultaneous infection of several hepatitis viruses; A variety of sexually transmitted infections, such as Neisseria gonorrhoeae, Treponema pallidum and HIV; Non-spore anaerobic bacteria infection requiring special culture; Bacterial infections such as tetanus, anthrax, Clostridium perfringens and Yersinia pestis caused by war wounds; (3) It is economical and simple, and can simultaneously detect multiple pathogens in the same reaction tube, saving reagents, cost and time, and providing faster, more and more accurate diagnostic information for clinic. Reyes et al. detected Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae from cerebrospinal fluid samples of 90 children with fever but negative culture by multiplex PCR, with specificity of 100% and sensitivity of 89%. Real-time fluorescence quantitative PCR, in which fluorescent groups are added to the PCR reaction system, and the whole PCR process is monitored in real time through the accumulation of fluorescent signals. It has the characteristics of high sensitivity, high specificity, effective solution to PCR pollution problem and high degree of automation. Real-time fluorescence quantitative PCR is of great value for early diagnosis, window screening, curative effect detection, gene variation analysis and prognosis evaluation of sexually transmitted diseases, which is helpful for epidemiological investigation. Wang Heng et al. established a multiplex real-time fluorescence quantitative PCR reaction system, and the same system can simultaneously and rapidly detect methicillin-resistant and enterotoxin A-producing Staphylococcus aureus. Specific primers labeled with fluorescent groups can accurately reflect pathogen infection and drug efficacy, and are especially suitable for the diagnosis of pathogens such as viruses and chlamydia, which cannot be cultured artificially and are difficult to culture. Gene chip technology is combined with multiplex PCR, and the target gene is amplified by PCR. The fluorescence probe of gene chip enhances the sensitivity and specificity of detection, which makes the advantages of the two detection technologies complementary, and has been widely used in the detection of pathogenic microorganisms. Primers and probes were designed with pathogen-specific genes as target genes, and oligonucleotide chips were prepared by multiplex PCR amplification. Then, the target gene of the sample to be detected is amplified by multiplex PCR, and the amplified product is hybridized with the pathogen multiplex PCR gene chip detection system. According to the hybridization signal, the species, types, virulence and invasiveness of pathogens contained in the sample can be intuitively interpreted, so as to detect and identify pathogens. (Progress in detection technology of pathogenic microorganisms) 3.4 Other gene detection technologies Molecular biology technology has developed rapidly, and various new gene detection methods are constantly emerging. In 2000, Notomi developed a new loop-mediated isothermal nucleic acid amplification method (LAMP). Four or six primers were designed for six or eight specific regions in the target gene sequence, and circular structure and strand displacement were formed under the action of DNA polymerase with strand displacement activity to amplify the target DNA in large quantities. In just a few years, LAMP has been widely used in disease diagnosis, food inspection, environmental monitoring, biological safety and so on. Mona Lee et al. used LAMP method to detect Clostridium tetanus in 16 cases of deep infection secretion of open wound for 60 min, among which 4 cases were positive, and the minimum detection limit was 4× 10. It is reported that 200 sputum samples of tuberculosis patients were quickly detected by LAMP technology, and the positive detection rate of Mycobacterium tuberculosis was much higher than that of culture method and staining method. MLVA (Multi-Locus Variable-Nun-Bertandem Repeat Analysis) is a genotyping technique based on the characteristics of variable number of tandem repeats in pathogen genome, which is widely used in genotyping and identification of Staphylococcus aureus, Escherichia coli and Bacillus anthracis. . Summary and prospect: rapid and accurate detection and identification of pathogens is the primary problem in the prevention and treatment of infectious diseases. With the development of biological research from macro-field to micro-field, the detection methods of pathogens have also gone deep from histomorphology level to molecular level and gene level. In recent years, the Qualcomm quantitative detection technology of pathogenic microorganisms has the advantages of less sample demand, fast and time-saving, no pollution, accurate diagnosis and high degree of automation. It is believed that with the continuous progress and deepening of research, these Qualcomm quantitative diagnosis techniques and methods will play an increasingly important role in the diagnosis and analysis of pathogenic microorganisms, and the combination and synthesis of various detection techniques will have broad application prospects.