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I'll give you some information about E. coli, which is rather messy. Go and tidy it yourself.
I have never heard of the term "three cold foods". If you can explain, maybe I will know.
Research progress in experimental diagnosis of Escherichia coli O 157: H7
Clinical manifestations of Escherichia coli O 157: H7 infection
Hemorrhagic enteritis: bloody stool, abdominal spasmodic pain, low or moderate fever.
Hemolytic uremic syndrome (HUS): It mainly occurs in children, and often occurs several days or 0-2 weeks after diarrhea. The mortality rate is generally 10%, which can be as high as 50% respectively.
Thrombotic thrombocytopenic purpura (TTP): It mainly occurs in adults, especially in the elderly. The main symptoms of patients are fever, thrombocytopenia, hemolytic anemia and abnormal renal function. The disease develops rapidly and the mortality rate is high, sometimes 70% patients die.
Infection source and transmission route of Escherichia coli O 157: H7
Escherichia coli O 157: H7 is basically a food-borne pathogen, which can be infected by eating beef, milk, beef or products, chicken, vegetables, fruits, drinks and water contaminated by Escherichia coli O 157: H7, and can also be spread through close contact between humans and animals.
In the laboratory, Escherichia coli O 157: H7 can infect mice, chickens, rabbits, pigs, cows and other animals.
The infection of Escherichia coli O 157: H7 has become a worldwide problem.
The situation in our country is also not optimistic.
I. Culture and Identification of Bacteria
1. Isolation and culture
Early culture is of great significance to identify pathogens.
The object of culture is acute bloody diarrhea, followed by HUS, TTP and other hospitalized patients, followed by high-risk contacts.
Medium:
Sorbitol Maikangkai agar, tryptone soybean agar
Improved eosin methylene blue and improved SD-39(MSD) agar.
Sorbitol MacKay agar (TC-SMAC) added with cefixime and potassium tellurite.
Sorbitol Maikangkai agar (CR-SMAC) added with cefixime and rhamnose (0.5%).
The chromogenic medium of Escherichia coli O 157: H7
Tetrabromofluoromethylene blue agar
H7 antiserum-sorbitol fermentation medium
Concentrated solution:
EB broth or intestinal enrichment liquid containing 10mg/L neomycin.
Peptone soybean broth containing10mg/l neomycin or10mg/l acridine hydrochloride.
Neomycin MEC broth
Lauryl tryptone broth
Adding any inhibitor in the following examples can increase the selectivity of the culture medium.
0.05 mg/L cefixime and 2.5 mg/L potassium tellurite.
Neomycin10mg/L Vancomycin 40mg/L.
2. Immunomagnetic bead separation method
Immunomagnetic bead separation method is to adsorb specific antibodies on adsorbable magnetic beads, and then enrich E.coli O 157: H7 in samples by using the reaction characteristics of antigen and antibody.
Methods: 1. Enriching bacteria; 2. Take the sample 1 ml and add 20 microliters of E.coli O 157: H7 immunomagnetic beads; 3. Gently stir 10 minute; 4. Insert the test tube into the magnetic frame; 5. Sucking the supernatant; 6. Add PBS and repeat the process of 3-5; 7. Take the sediment from the tube wall, line it and inoculate it in selective medium, such as CT- sorbitol MacKay agar (C- cefixime, T- potassium tellurite). Culture at 37℃ for 6-24 hours, and select suspicious colonies for identification.
Chapman et al. * * * detected 690 fecal samples, 25 of which were detected by immunomagnetic bead separation. Among the Escherichia coli O 157: H7 isolated by immunomagnetic beads, only 5 strains were positive by direct culture.
Wright et al. cultured the beef minced samples inoculated with E.coli O 157: H7 in buffered peptone water containing vancomycin and cefixime, and then isolated the bacteria directly or with magnetic beads coated with E.coli O 157 antibody, and inoculated them into CT-SMAC. As a result, the detection amount of direct secondary culture is 200cfu/g, while the immunomagnetic bead separation method only needs 2 cfu/g. ..
Chapman et al. first enriched bacteria with EC broth, then enriched E.coli O 157: H7 in bovine feces by immunomagnetic separation, and then separated it with selective culture medium, and compared it with CR-SMAC and CT-MAC direct culture methods. The former is 12 strain of Escherichia coli O 157: H7, and its sensitivity is 100 times higher than that of direct culture on two kinds of media.
Cubbon et al. detected E.coli O 157: H7 in cattle feces and food samples by immune magnetic separation (IMS). In an outbreak of milk-borne Escherichia coli O 157: H7, IMS, fecal culture and polymerase chain reaction were used to detect Vero toxin gene carriers, and the isolation rates of fecal Escherichia coli O 157: H7 were compared among the three methods. Of the 142 fecal samples examined, 20 were positive for direct culture and IMS. Therefore, IMS improved the detection rate of E.coli O 157: H7 infection cases, and the coincidence rate with PCR was high.
At present, contaminated meat, livestock and even drinking water are threatened by Escherichia coli O 157: H7. Traditional culture methods are not ideal for detecting intestinal pathogens in food and water. The study of IMS and other detection methods shows that the rapid detection of food and environmental samples seems to have a good prospect. Ion mobility spectrometry combined with electrochemiluminescence detection (ECL) was used to detect Escherichia coli in food and pollutants. The results showed that the detection standard of Escherichia coli in the original buffer was 100- 1000 cfu/lm, and the detection range in food was 1000-2000 cfu/lm, and the detection time was very fast, generally less than 1 hour. The sensitivity of cow dung suspension of strain O 157: H7 on two kinds of culture media is 100 times higher than that of direct culture. During the four-month monitoring period, a total of 24 samples of bovine rectum 1024 were collected, among which Escherichia coli O 157: H784 (8.2%) was detected, and 23 samples (27.4%) were isolated by direct culture and IMS. In 23 samples, 15 was separated by two media, of which 5 were separated by CT-SMAC, 3 by Cr-SMAC, and the remaining 6 1 (72.6%) was separated by IMS. Washing magnetic beads with PBS containing Tween -20 can reduce the nonspecific binding of other microorganisms to magnetic beads. Escherichia coli O 157: H7 will not bind when the magnet is coated with irrelevant antibody. IMS is rapid, simple and specific, and has certain value in epidemiological investigation.
3. Biochemical reaction
At present, O 157 and H7 specific antibodies used in many countries have different degrees of cross-reaction with a few bacteria. Therefore, it is necessary to identify E.coli through biochemical tests.
The main biochemical reaction results of typical Escherichia coli are as follows:
Kinetic test (+) glucose (+) maltose (+) mannitol (+) sucrose (-/+) hydrogen sulfide (-) urea (-) indigo substrate (+) methyl red (+) V-P test (-) citrate (-) phenylalanine decarboxylase (-) lysine decarboxylase (+/-
Please refer to table 1 for biochemical reactions that are significant for O 157 identification.
MUG is 4- methyl umbelliferone-β-glucuronide. Most Escherichia coli have glucuronidase, which can hydrolyze MUG to produce fluorescence, but most strains in O 157: H7 do not hydrolyze MUG. Thompson et al. established a rapid MUG test, in which 0.5 ml of MUG reagent was placed in a test tube, the pure culture of the bacteria to be detected was picked up with sterile cotton swabs and mixed evenly, and observed in a dark room at 44.5℃ for 20 minutes under high-intensity light source, and the results were obtained. Those that produce blue fluorescence are positive.
Two. Serological test
1. Slide agglutination test
2. Colloidal gold immunoassay
3. Enzyme-linked immunosorbent assay
4. Immunofluorescence technology
5. latex agglutination
6. Indirect hemagglutination analysis (IEHA)
7. Automatic antigen-antibody detection system
8. Western blot
1. Slide agglutination test
Slide agglutination test is the most classic method to identify O antigen. If the agglutination reaction is not clear in the experiment, but the clinical manifestations and biochemical reaction strains are highly suspicious, the bacterial liquid can be heated at 100℃ for 30min and then slide agglutination test can be carried out. This can remove the influence of K antigen. In order to eliminate the false positive caused by agglutination reaction caused by cross reaction, tube agglutination reaction should be done, and the titer determined should not be less than half of the original calibration titer of diagnostic serum. To identify H antigen, both slide test and tube agglutination test should be done at the same time. The bacteria to be detected should be done first, and the culture should be taken for H antigen identification when power is applied.
2. Colloidal gold immunoassay
Colloidal gold immunoassay is characterized by antigen-antibody reaction with colloidal gold as a marker. This technique was originally used for immunoelectron microscopy. Up to now, in immunoassay, gold labels often cooperate with membrane carriers to form specific patterns, such as dot immunofiltration test and dot immunochromatography test. As a simple and rapid serological detection method, gold labels have been widely used.
Colloidal gold is mostly prepared by reduction method, and chloroauric acid is the main reducing substance. The size of gold particles depends on the amount of trisodium citrate added in the preparation process.
In the colloidal gold immunochromatography test, nitrocellulose membrane is used as the carrier, and the liquid dripping at one end of the membrane is slowly transferred to the other end by capillary action of microporous membrane, just like chromatography.
The Institute of Microbiology, Chinese Academy of Preventive Medicine has developed a gold card for rapid detection of Escherichia coli O 157: H7 pathogens by using colloidal gold technology, double antibody sandwich method and color reaction, which is generally used for qualitative detection of Escherichia coli O 157: H7 in feces, food, water and other samples. The degree of color development is directly proportional to the bacterial content in the sample, and the minimum bacterial detection amount is less than 100 bacterial cells. Its main feature is high sensitivity, which can be used for the initial screening of samples to be tested, and positive samples can be separated by bacteria, reducing workload.
3. Enzyme-linked immunosorbent assay
The common detection method of Escherichia coli O 157: H7 is to isolate bacteria after fecal culture, and then identify them by biochemical and immunological methods, which generally takes 72 hours.
Dylla et al. directly detected E.coli O 157: H7 in feces by rapid ELISA, and compared with the standard culture method. The results showed that 9 strains of Escherichia coli O157: H7 were detected in 83 fecal samples by ELISA. Conventional culture was negative for 176, while ELISA was negative for 174. The overall specificity was 98.9%. This method has no cross reaction with other non-O 157: H7 Escherichia coli, and is an accurate, sensitive, specific and easy-to-observe screening method, especially suitable for epidemiological investigation of small and medium-sized laboratories and a large number of fecal samples.
Padhye et al. detected O 157: H7 by direct ELISA reaction with monoclonal antibody, and found no cross reaction with Salmonella, Yersinia colonitis, Shigella and Klebsiella pneumoniae except O26 :H 1 1+0. Monoclonal antibody has obvious specificity in detecting Escherichia coli O 157: H7, and can be used as an immune reagent for rapid detection of clinical and food samples. Clark's further study of monoclonal antibodies found that the substance recognized by this monoclonal antibody is LPS. This LPS antigenic determinant can also be detected by whole-cell ELISA in other serotypes of Escherichia coli and Escherichia coli with or without Vero toxin, and is easily affected by bile salts, acridine yellow and temperature. However, the modification scheme combined with immune capture can improve the specificity of ELISA
4. Immunofluorescence technology
Skillful;
The main feature of direct fluorescence technique (DEFT) and antibody-based fluorescence technique (AB-DEFT) is to directly count bacterial cells in samples under a microscope. Because there is no need for culture or separation process, the detection is very fast. The basic principle of DEFT is to filter the sample, dye the bacterial cells left on the filter with fluorescent dyes (such as acridine orange), and then observe and count them with a fluorescent microscope. However, due to the nonspecific staining of fluorescent dyes, not only the tested bacteria but also the background bacteria will be stained, which will affect the accuracy of the results. Ab-DEFT overcomes this shortcoming, and the filtered bacterial cells interact with the specific antibody labeled with fluorescence, and then are observed and counted by fluorescence microscope.
Solid-phase fluorescence capillary immunoassay
This method has the advantages of high sensitivity, rapidness, less reagent consumption and simple operation. Czajlu et al. used thermally killed O 157: H7 bacteria to coat glass capillaries as solid supports.
The polyclonal antibody O 157: H7 combined with biotin was added to the sample for a period of time, then the sample/antibody mixture and avidin labeled with Cy5 (a fluorescent cyanine dye) were added to the capillary, incubated for 2 minutes, washed and dried, and the cyanine dye was excited by the excitation light with the wavelength of 650nm emitted by the laser sensor system, and then the fluorescence emission density was measured by the optical sensor system.
Direct immunofluorescence antibody staining
Park et al. centrifuged stool samples and labeled stool smears with immunofluorescent antibodies, all strains confirmed by culture method could be detected, and the detection time was.
5. latex agglutination
In this method, latex particles were used as carriers and sensitized with specific antibody O 157: H7 to prepare specific latex reagent. The specimen was emulsified on a glass slide or a colored burning plate, and latex reagent was added dropwise. When it was obviously agglutinated, it was positive when the control latex was not agglutinated.
6. Indirect hemagglutination analysis (IEHA)
This method is mostly used for antibody detection of LPS, soluble large antigen and unheated antigen, but it is ineffective for antibody detection of H antigen. Morooka et al. detected the serum LPS antibody of patients with hemolytic uremic syndrome (HUS). Although formaldehyde sheep red blood cells (SRBC) have a low level of nonspecific adsorption, this does not affect this method as an effective and rapid (
7. Automatic antigen-antibody detection system
VIDAS is an automatic antigen-antibody detection system. It can directly detect the antigens, antibodies or toxins of bacteria, viruses, Toxoplasma gondii, chlamydia, spirochetes and other microorganisms from patients with infectious diseases.
Basic principle: Using the principle of enzyme-linked immunosorbent assay (sandwich method), the substrate was doped with fluorescent substances, and finally the fluorescent product 4- methyl -7- hydroxy concanavalin was produced. The fluorescence intensity is related to the concentration of the detected substance in the sample. Calculate the standard value by comparing the reading of the scanned sample with the standard, and judge the result according to the positive and negative critical values.
8. Western blot
Objective To detect the specific antibodies against lipopolysaccharide and hemolysin in sera of patients infected with E.coli O 157: H7. The basic principle is to separate and purify lipopolysaccharide or hemolysin by SDS- PAGE, then transfer it to nitrocellulose membrane by transfer electrophoresis, and then use antigen and antibody for immunoassay. Including SDS- polyacrylamide gel electrophoresis, transfer electrophoresis and immunoassay.
It is characterized by high specificity and sensitivity.
Immune detection
Cut the sealed nitrocellulose membrane into a swimming lane according to the width of comb teeth and number it in turn.
1) Add 1 ml patient serum diluted with PBS to each piece, shake it evenly at room temperature for 2 hours, and set up positive control and negative control;
2) Rinse with 2.5 ml /PBST for 3 times, 5 minutes each time;
3) adding II antibody diluted with 1 ml PBS, and shaking at room temperature 1 hour;
4) Wash it twice with 2.5 ml/PBST for 5 minutes each time, and then wash it again with PBS;
5) put the membrane into 12 ml chromogenic solution to develop color, and shake it at room temperature for 15-30 minutes until the positive control appears satisfactory blue-purple;
6) put the film into distilled water to stop the color reaction and take pictures;
7) When the color band is consistent with the molecular weight of hemolysin or the band spectrum of 0 157 lipopolysaccharide, the judgment result is positive.
Two. Molecular biological detection
The emergence of molecular biology provides many molecular tools for diagnosing microorganisms more quickly. This new study uses genotype rather than phenotypic factors to identify specific pathogens. Therefore, its specificity is better. Coupled with the automation of operating procedures, DNA analysis has become a routine operation in laboratory applications. Automatic DNA extractor, polymerase chain reaction instrument (PCR instrument), DNA sequence analyzer and pulse gel electrophoresis instrument (used to separate large DNA fragments, such as complete chromosomes) are very useful in disease diagnosis.
1.DNA probe
Probe labeling: radionuclide and non-radioactive substance labeling.
Source of probe: ① cloned genomic DNA probe; ②cDNA probe; 8 8 ⑧RNA probe; ④ Oligonucleotide probe.
Specimen treatment: different treatments are carried out according to the source and quantity of specimens.
Hybridization methods: dot hybridization, southern hybridization, in-situ hybridization, Northern hybridization, etc.
Detection of hybridization signal: (1) measure the number of radionuclide ray pulses (2) autoradiography (3) chromogenic method (4) luminescence method.
The slt and Ⅱ genes on o 157:H7 specific phage, the large plasmid hemolysin gene and eae gene on chromosome have all been made into specific probes for hybridization detection. Beutin et al. used probes such as VT 1(750bp), VT2(850bp) and hemolysin (3.4kb) to carry out epidemiological investigation. Thomas et al. detected different phage types O 157: H7 with digoxin-labeled VT2B subunit gene and VT2C gene probe. Huck et al. screened the restriction fragment on the large plasmid O 157: H7 and developed a 2.0kb Sma I fragment probe, which is considered to be the most specific for serotype O 157: H7 and can hybridize with all experimental bacteria O 157: H7.
2.PCR technology
Polymerase chain reaction (PCR) is a method to selectively amplify DNA or RNA fragments in vitro. It has the advantages of strong specificity, high sensitivity, rapidity, simplicity, amplification of RNA or cDNA, and low requirements on the quality of starting materials.
Basic components of PCR amplification system
Primers: The specificity of PCR products mainly depends on the specificity of primer chains. Due to the existence of homologous sequences and the random design of primer chains, multiple chains may appear in PCR products during electrophoresis analysis, so primer specificity should be fully considered when designing primer chains. The length of the primer is generally 15-30 bases, the content of G+C is 40- 60%, and the concentration is 0.1-1μ mol/L. ..
TaqDNA polymerase: the concentration is 1-4ul/ 100ul. The increase of unit dose of TaqDNA polymerase may lead to nonspecific DNA amplification.
Template DNA: Any protease, nuclease, DNA polymerase inhibitor and protease that can bind DNA should be avoided. The preparation methods of DNA template include heating method, freezing method, ultrasonic crushing method, alkali denaturation method, SDS cracking method and so on.
4× dntps: the pH value of dntp storage solution should be 7.0. In the reaction system, the concentrations of the four DNTP should be the same, and the concentration of each DNTP should be 50-200 umol/L.
Buffer and other components: Tris-HCl buffer is generally used in PCR reaction system. The suitable concentration of Mg2+ is 0.2-2.5 mmol/L higher than the total concentration of dNTP.
Cyclic parameters of PCR technology
PCR amplification is realized by repeated cycles of denaturation, annealing and extension. Determining the correct cycle parameters is the guarantee for the success of PCR.
Cyclic parameter
1. denaturation temperature and time
Incomplete denaturation of template DNA and PCR products is one of the most common reasons for the failure of PCR reaction. In the denaturation step, the denaturation condition is that the double-stranded DNA is completely separated at 95℃ for 30 s. For the target DNA sequence with high GC content, higher denaturation temperature is suitable. At the melting temperature, DNA denaturation takes only a few seconds. However, it takes some time to reach the melting temperature in the reaction tube. In principle, the denaturation step should be high temperature and short time, that is, sufficient denaturation should be ensured and the activity of polymerase should be maintained throughout the reaction.
Cyclic parameter
2. Primer annealing
The temperature and time of primer annealing depend on the length of primer, base composition and its concentration in the reaction system. For a typical oligonucleotide primer with GC content of about 50% and length of 20 bases, the optimal annealing temperature is 55℃. High temperature annealing can improve the specificity of PCR.
Cyclic parameter
3. Primer extension: Primer extension means that DNA polymerase adds deoxymononucleotide to the 3'-OH end of primer one by one, and the primer extension temperature depends on the optimal temperature of DNA polymerase. If TaqDNA polymerase is used, 70-75℃ and 72℃ are generally used. The time of the extension step depends on the length, concentration and extension temperature of the target sequence. The longer the target sequence, the lower the concentration, the lower the extension temperature and the longer the extension time. On the contrary, the shorter the target sequence, the higher the concentration, the higher the extension temperature and the shorter the required extension time. Generally speaking, for every 1000 base sequence, the extension time of 1 min is enough. For the amplification of short sequence fragments of 100-300 bases, the step of temperature extension can be omitted and a fast and simple double temperature cycle of denaturation and annealing can be adopted. This is because TaqDNA polymerase maintains very high activity even at annealing temperature, and the extension process can be completed when annealing temperature becomes denaturation temperature.
Cyclic parameter
The length of the amplified product is100bp500bp1000bp2000bp.
Degeneration time at 94℃ (seconds) 30 30 60 60
Renaturation time at 55℃ (seconds) 30 30 60 60
72℃ extension time (seconds) 30 38 120 180
Generally, 25-30 cycles are enough, and the last cycle time and extension time are increased by 5 minutes.
Detection method of PCR amplification products
After circulating amplification of PCR reaction mixture, what needs to be done is to detect whether the expected amplification products and product specificity exist in the reaction solution. At present, many methods for detecting and analyzing PCR amplification products have been developed. Include gel electrophoresis, high pressure liquid chromatography, nucleic acid probe hybridization, probe capture enzyme immunoassay, restriction map analysis, single strand conformation polymorphism analysis and nucleic acid sequence analysis.
PCR technology type
ImmunoPCR, in situ PCR and asymmetric PCR.
Nested PCR technology reverse PCR technology reverse transcription PCR technology
Compound PCR, color PCR and antigen capture PCR.
Sensitization PCR technology, enzyme-labeled PCR technology and two-temperature PCR technology
Anchor PCR, quantitative PCR, capillary PCR
Multiplex PCR technology nested or nested PCR technology
Application of PCR in detection of Escherichia coli O 157: H7
(1) Simple PCR: Meng et al. designed a pair of primers based on a 688bp DNA fragment near the 5' end of eae gene, and the amplified product was a 633bp DNA fragment. The annealing temperature is 60℃-63℃. The detection limits of Escherichia coli O 157: H7 by boiling method and gene release method were 25 and 38CFU/ml, respectively, and the detection time was 3 hours.
Thomas et al. amplified slt gene fragment by PCR. Primer:
Positive chain 5'-(tttacgatagatttctgaccc)-3',
Reverse chain 5'-(cacataaatttttcgctc)-3'
PCR products were determined by gel electrophoresis, and the detection time was ld.
Xu Jianguo et al. designed PCR primers according to the unique hlyA and b gene sequences of O 157: H7, and the product was 338bp.
Application of PCR in detection of Escherichia coli O 157: H7
(2) Multiplex PCR: Because the identification of serotype O 157: H7 cannot rely solely on simple PCR, in recent years, foreign scholars have studied the diagnostic value of multiplex PCR for Escherichia coli O 157: H7. Meng et al. simultaneously amplified eae upstream gene fragment, SIT Ⅰ gene fragment and SIT Ⅱ gene fragment, the lengths of which were 633, 2 10 and 484bp respectively. The primer design can effectively distinguish O 157: H7 serotypes from O55: H7 and O55: NM serotypes. Fratamico et al. simultaneously amplified eae gene, conserved sequences of slt and Ⅱ and conserved sequences of 60MDa plasmid in one reaction, and the products were 1087, 227, 224, 166bp respectively. Yan Li selected three pairs of primers for Shiga-like toxin Ⅰ, Ⅱ (SLT-Ⅰ, SLT-Ⅱ) and hemolysin (Hly) genes of Escherichia coli O 157: H7, and carried out PCR in the same amplification system to detect Escherichia coli O 12 strain and other pathogenic Escherichia coli and Salmonella. Results Multiplex PCR was more specific than single PCR, and the detection results of strain O 12+057: H7 were stable and reliable. It can be quickly and effectively distinguished from other pathogenic Escherichia coli, Salmonella and Shigella.
Application of PCR in detection of Escherichia coli O 157: H7
(3) In-situ PCR:kurokawa et al. used in-situ PCR technology combined with emission microscope to quickly detect O 157: H7 at the single cell level without culture process.
4.23 Application of SRRNA in typing and detection of Escherichia coli O 157: H7.
The traditional classification methods of bacteria mainly depend on the characteristics of bacteria, such as morphology, metabolites, enzyme activity and surface antigen. With the rapid development of modern molecular biology theory and technology, microbial detection has entered the gene era, and the classification method based on ribosomal RNA sequence provides a new molecular biology method for microbial identification. Such as 16srRNA, 23srRNA, 16-23srRNA interval sequence analysis, etc. It is completely different from the traditional method and has the advantages of rapidity, simplicity, sensitivity and specificity.
Characteristics of 23SrRNA gene:
Prokaryote ribosomes have three sizes (23S, 16S, 5S). At present, there are 250 strains with the full-length sequence of 23SrRNA gene. The length is about 3000pb. The sequence analysis of 23SrRNA gene of many bacteria shows that the variability of its sequence is more obvious than that of 16SrRNA gene, especially those bacteria with close genetic relationship. Using the differences of these variable region sequences, different strains of the same strain can be classified and identified. At the same time, the sequence analysis of the known 23SrRNA gene also found that there were six conserved regions (5- 10 region) in the initial 520pb, and it was found that the 6 and 10 regions of these six conserved regions were the most conserved, and the sequences were completely consistent among 14 strains.
Application:
In order to detect the pathogens of clinical infectious diseases, two primers were designed as universal primers in the conservative region, and the sequence was selected as a specific probe in the variation region. First, PCR amplification was carried out with universal primers to screen out samples containing pathogens, and then specific probes were used to hybridize with the amplified products to identify the target bacteria, thus achieving the purpose of diagnosing pathogens and typing.
Identification of specific bacterial species At present, it is considered that IVSs of 23SrRNA gene is species-specific, and PCR amplification with IVSs (insertion sequence) can realize the diagnosis of a certain genus of bacteria.
The application in epidemiology can use the difference of variable region sequence to analyze different strains of the same strain. Provide evidence for the epidemic.
In addition to the above methods, the commonly used effective detection methods include pulsed field electrophoresis, random amplified polymorphic DNA fingerprinting, cytotoxicity test and so on. I won't go into details here.
Test procedure for Escherichia coli O 157: H7
The sample was enriched for 6 hours and concentrated with magnetic beads.
Suspicious colony sorbitol Maconchy agar
G staining biochemical reaction serological virulence gene
Laboratory diagnosis of Escherichia coli O 157: H7
One of the following conditions has the significance of laboratory diagnosis:
1) Escherichia coli O157: H7 was isolated from stool samples of diarrhea patients;
2) It is confirmed by PCR or DNA hybridization test that it has hemolysin gene and Shiga-like toxin gene;
3) The serum anti-O 157LPS IgG antibody of diarrhea patients increased 4 times in the recovery period;
4) The acute serum or convalescent serum of patients with bloody stool diarrhea was confirmed to contain specific antibodies against O 157LPS, EHEC hemolysin or Shiga toxin by protein blot test.
summary
Since O 157: H7 was recognized, its genes have been studied more and more carefully, and many structural and functional genes have been identified, which has greatly promoted its etiology, pathology and clinical treatment. Because the dose of O 157: H7 is very low, it is necessary to develop some highly sensitive methods for rapid and effective detection. In addition, there are many mutant strains, and it is often not enough to detect them by one method alone. For example, SMAC could not detect the mutant of fermented sorbitol; Because many non-EHEC(EPEC, Vibrio cholerae, Shigella, etc. ) can also produce SLT, and simple detection of SLT can also produce false positive results. Therefore, the detection of a sample needs to combine multiple methods to obtain accurate results.
Chapter III Determination of Coliform Group
A, coliform bacteria test
(1) test method
(2) culture medium
(III) Matters needing attention in inspection
Second, the hygienic significance of coliform bacteria
Coliform group is one of the important indexes to evaluate the quality of food hygiene, which has been widely used in food hygiene at home and abroad. This flora mainly comes from the feces of human beings and warm-blooded animals, and is generally used as an indicator of fecal pollution in food. In the past, there was not much experience in detecting coliform bacteria in China, and the understanding of this flora was not sufficient. 1974 national symposium on revision of inspection methods of food hygiene bacteria and 1976 national food hygiene standards conference suggested that coliform bacteria should be used as indicator bacteria of fecal pollution, and put forward scientific research work of coliform bacteria. Therefore, we set up a scientific cooperation group for coliform bacteria, and conducted extensive scientific research and practice on the detection methods (including rapid detection methods) of dog intestinal flora and its hygienic significance, and achieved certain results, which provided a scientific basis for formulating the detection methods of coliform bacteria.
Coliform group In the process of revising the l976 edition of Food Hygiene Inspection Methods, the coliform group inspection methods in 1983 ~ 1985 were studied experimentally and compared. At the same time, the common rapid detection methods of coliform bacteria in China were discussed, which provided scientific basis for the determination of microbial coliform bacteria in the revised national standard "Food Hygiene Inspection Methods".
Coliform bacteria are not classified and named in bacteriology, but a group of bacteria related to fecal pollution according to hygienic requirements. These bacteria are not completely consistent in biochemistry and serology. Its definition is: refers to a group of aerobic and facultative anaerobic, which can decompose lactose at 37℃ to produce acid and gas. Some scientists use indigo matrix, methyl red, V ~ P, citrate, hydrogen sulfide, gelatin, power and lactose to decompose at 44.5℃, and divide this group of bacteria into Escherichia coli, Citrobacter, Klebsiella aerogenes and Enterobacter cloacae. No matter what classification method is adopted, the determination of coliforms should be based on the above definition.
A, coliform bacteria test
(1) test method
1. Lactose fermentation test. Take samples by aseptic operation, and determine the dosage and dilution multiple according to the requirements of national or local health standards and the pollution of samples. Inoculate the sample to be tested into a lactose bile salt fermentation tube. If the inoculation amount is greater than l m J, use a double lactose bile salt fermentation tube, and if l m is less than 1 and 1mI, use a single lactose bile salt fermentation tube. Inoculate 3 tubes at each dilution, put them in an incubator at 36+1℃, and cultivate for 24+/-2 hours. If all lactose bile salt fermentation tubes do not produce gas, they can be reported as negative for coliform bacteria. If gas is generated, follow the following procedure.
2. Isolation and culture. Transfer the gas-producing fermentation tube to eosin methylene blue agar plate, put it in an incubator at 36 L℃ for 65438 08 ~ 24 hours, then take it out, observe the colony morphology, and carry out Gram staining and confirmation test.
3. Confirm the test. On the above plate, l ~ 2 suspected coliforms were selected for Gram staining. At the same time,