In recent years, with the rapid development of bovine molecular biology technology, new diagnostic techniques and methods have emerged and been widely used in the detection of clinical microorganisms, providing important information for the analysis of pathogenicity, prevalence, variability and drug resistance of pathogenic microorganisms. At present, the methods of typing pathogenic mini-cattle by molecular biology technology are: pulsed field gel electrophoresis. PFGE) typing, polymerase chain reaction (PCR) typing, biochip typing and multiple sequencing typing (MI). ST), plasmid DNA pattern typing and restriction fragment length polymorphism (RFLP) typing.
1. PFGE typing technology of pulsed field gel electrophoresis is known as the "gold standard" for bacterial molecular typing because of its good repeatability and strong resolution. It can be used to separate macromolecular DNA with a resolution range of 10 Mb, while ordinary agarose gel electrophoresis can only separate DNA less than 500 Kb. The basic principle of PFGE is that the swimming direction of DNA molecules embedded in gel is formed by the constant change of electric field.
Biologically, small molecular DNA swims faster than large molecular DNA, and a specific electrophoresis pattern is displayed on the gel of [ⅱ] according to the size of DNA molecules. Pulsed field gel electrophoresis can effectively isolate the genomic DNA of pathogenic microorganisms. The density of DNA bands reflects the genomic DNA content and molecular size of pathogenic microorganisms, and finally achieves the purpose of typing. At present, PFGE has been widely used in the typing of pathogenic mini-cattle. Swaminathan et al. ∽J established the standard PFGE operation method for typing pathogenic microorganisms, such as Escherichia coli 0 157: H7, Salmonella typhimurium serotype, Listeria and Shigella. Obtain research recognition
The resolution of PFGE is better than that of ribosome typing and random amplified polymorphic DNA (RAPD) typing. When the resolution of 1 restriction element is not strong, two kinds of restriction enzymes can be used to improve it. Of course, PFGE also has some limitations, such as long time-consuming and high cost. In addition, electrophoresis patterns are easily influenced by the technical level of operators and other factors, which makes it difficult for different laboratories to compare rice.
2. Since the invention of polymerase chain reaction in 1985, people have attached great importance to it because of its high sensitivity and specificity, and it has become a routine method for nucleic acid amplification and detection. There are two main PCR methods for molecular typing of pathogenic microorganisms: RAPD typing and repetitive sequence PCR ploughing. The RAPD based on PCR is 1 gene, which can be used for the whole unknown sequence.
Molecular biological methods for polymorphism analysis. In this method, kevin keegan DNA was used as a template, and random polymorphic nucleotide sequence (usually 10 base) synthesized by a single person T. was used as a primer, and PCR amplification was carried out under the action of heat-resistant DNA polymerase. The polymorphism of the amplified products was analyzed after agarose or polyene amide gel electrophoresis. This reaction is similar to the characteristics of genomic DNA, thus classifying pathogenic microorganisms. RAPD can analyze the DNA polymorphism of species without any genomic information, and the purity of template DNA is not high. DNA probes and molecular hybridization are not needed. PCR typing of repeated sequences of 1996
1 The method of bacterial genome fingerprinting, that is, PCR amplification of short repetitive sequences widely distributed in bacterial genome, is described in, and the differences between genomes are revealed by comparative analysis of electrophoresis patterns [5]. Studies have shown that PCR typing of repeated sequences has the same resolution as RAPD typing [6], but the operation is relatively complicated. However, the repeatability of PCR typing of repeated sequences is very good, which is incomparable to RAPD. In addition, multiplex PCR, nested PCR, etc. It is also used for the classification of pathogenic microorganisms. Although each has its own advantages, it also has some shortcomings, such as weak resolution, poor repeatability and difficult analysis of results, so it has not been widely used in clinic.
3. Biochip typing Biochip technology is to fix biomacromolecules, such as oligonucleotides, cDNA, kevin keegan DNA, peptides, antigens, antibodies, etc., on solid media such as silicon wafers, glass wafers, plastic wafers, gels, nylon membranes, etc., to form a biomolecular lattice. When the biomolecules in the sample to be detected hybridize or interact with the probe molecules of the biochip, the hybridization signal is detected and analyzed by the J laser * * * focusing microscope scanner. The basic principle of its application in pathogenic microorganism typing is to make specific genes representing each subtype into 1 chip, and the subtypes of pathogenic microorganisms in samples can be detected and identified by reverse transcription. Suspended Chip Technology (SAT), also known as Microglobulin Chip (PBA), is a new chip technology of 1 which appeared in recent years. The principle is that the microspheres with a diameter of 5.6 ttm are dyed into 100 colors by throwing two fluorescent dyes in different proportions.
Color, each color of microspheres * * * valence binding 1 breeding cattle probe, which can be antigen, antibody, ligand, nucleic acid or enzyme, respectively targeting 1 species to be detected. By mixing microspheres loaded with 100 different colors, 1 00 different biological reactions can be completed simultaneously in1reaction well. Then the microspheres pass through two pipelines irradiated by laser beams in a single file, and the computer can collect and process the fluorescence intensity changes of each color microsphere, and carry out qualitative or quantitative detection on each object. The system is used for the joint detection of various microbial antigens, antibodies and specific genes. At present, this method has been applied to clinical detection of human papillomavirus typing. Compared with solid-state chips, liquid chips are the opposite.
It has great advantages in kinetics, reaction speed, detection sensitivity, stability and automation, so many scholars are optimistic about the application prospect of liquid-phase chips.
4. Multi-locus sequence typing With the rapid development of DNA sequencing technology, molecular typing tends to be single or multiple loci polymorphism of chromosomes. Mi ST typing refers to determining the nucleotide sequence of the core fragment with a length of about 470 bp in several housekeeping bases, and indexing their combinations. Different strains correspond to different sequence types, thus revealing the diversity of alleles among strains. Maid-en et al [83] found that MI and ST can be used for typing Neisseria meningitidis. They think that the sequence analysis and comparison of several housekeeping genes have achieved a balance between the II I operability in the experimental process and the reliability of the results, and the results are accurate. The obtained data have good comparability in different experiments, that is, they have strong intra-species resolution for a certain strain. 9j. Chen et al. no .] was used to carry out genetics on 5 1 Candida albicans isolated from Taiwan Province 12 Hospital in China.
Results 83 polymorphic loci and 45 diploid sequence types were found in 7 housekeeping gene sequences. Among them, 36. 1% is synonymous mutation and 63.9OA is non-synonymous mutation. They think that I ... ST has a stronger resolution than PFGE, which can distinguish patients' feelings.
Micro-intraspecific evolution of Candida albicans over time. However, the disadvantages of MLST are its high cost and special instruments needed in the operation process. This makes this technology limited to large global epidemiological research centers.
Use, affect its popularity in hospitals.
5. Plasmid DNA mapping Bacterial plasmid analysis is an early molecular typing method for pathogenic mini-cattle. The method includes plasmid DNA extraction and agarose gel electrophoresis. Because different strains have different plasmid DNA sequences and sizes,
The DNA plasmid profiles separated by agarose gel electrophoresis are also different, so different strains can be typed. The more plasmids a strain carries, the stronger the specificity of plasmid DNA mapping method. The advantages of plasmid network typing are relatively simple operation, short time consumption, low cost and simple equipment. However, the plasmid map has an insurmountable defect. That is, plasmids can be lost, acquired and transferred spontaneously between the same kind of bacteria or even between different kinds of bacteria, resulting in unstable plasmid maps. In addition, plasmid mapping method can not distinguish those plasmids with the same size but different DNA sequences.
6. RFI typing of restriction fragment length polymorphism. P means that genomic DNA is digested by restriction enzymes, and the digested fragments are separated by agarose gel electrophoresis. A large number of short fragments can be produced by digesting the genomic DNA of pathogenic microorganisms with restriction endonucleases Bgl and EcoRI, and the DNA map obtained by electrophoresis can be used for typing pathogenic microorganisms. almost everywhere
Dumachi, a pathogenic bovine isolate, was typed by this method. However, due to the huge genome DNA, there are many fragments produced after enzyme digestion, and there are a lot of overlapping fragments, which makes it difficult to use the consistency analysis surface I of the map between Wei plants. RFLP score
The resolution of type A is weaker than that of PFGE, and the operation is more complicated.