(1) National standard method (conventional method)
1. Enrichment culture
2. Isolation
3. Identification
p>(2) Gold label test paper method
This method is the “Emergency Treatment of Enterohemorrhagic Escherichia coli O157:H7 Infectious Diarrhea Nationwide” published by the People’s Republic of China and the Ministry of Health. specified method in the plan.
After the sample has undergone special pre-enrichment and enrichment, take 120ul of the enrichment culture solution and put it into the sample tank of the test paper clip. The sample moves to the reaction area due to capillary action. The reaction area contains special The antibody (E.coliO157:H7 antibody) is conjugated to colloidal gold particles. If there is an antigen in the sample, it will combine with the gold-labeled antibody to form an antigen-antibody complex, and move forward along the nitrocellulose membrane to the area (T area) containing the fixed anti-E.coliO157:H7 antibody. The complex of the gold-labeled immune The substance specifically binds to the anti-antibodies in this region to form a visible line. Other samples continue to move to the end of the membrane, eventually entering the waste pool and being discarded.
The reaction zone also contains a patented antigen (color indicator) bound to a gold label, which is eluted by the sample regardless of the presence or absence of the E.coliO157:H7 antigen in the sample. The gold-labeled control indicator moves through the membrane to the negative control zone and binds to the patented antibody to form a visible line. Regardless of whether there is E.coliO157:H7 antigen in the sample, the control line will be formed in the control area (C area) to ensure that the test proceeds normally.
After 8-10 minutes of instillation of the enrichment solution, observe whether there are obvious lines in the C and T areas of the test paper. If there are visible lines in both the C and T zones, the result is positive. If area C is wired and area T is wireless, the result will be negative. If area C is wireless, regardless of whether there is a line in area T, the experiment will be invalid.
(3) ISO-GRID detection system
The ISO-GRID detection system is a filtration system based on hydrophobic mesh membrane (HGMF). The system detects or counts microorganisms by using a filter membrane containing 1,600 small squares.
First, the diluted sample is filtered through a 5um stainless steel pre-filter to filter out sample residue particles that may cause errors in microbial analysis.
Then, the sample is filtered through a membrane filter in running water. The filter membrane is then placed on a specific agar culture plate and cultured to detect target microorganisms. After the incubation is complete, the target microorganisms are detected on the membrane and the number of positive areas is recorded.
The filter membrane will not be stained by the color of the agar medium, making it easy to distinguish, identify and count microorganisms. This method is fast, simple and reproducible. In addition to E. coli O157:H7, it is also suitable for the detection and enumeration of salmonella, yeast, mold, coliforms and E. coli.
(4) Monoclonal enzyme-linked immunoassay screening method
This method is to screen for the presence of E.coliO157:H7 in all foods. It is not a confirmatory test because the Monoclonal antibodies may cross-react with a small number of non-E.coli O157:H7.
Positive quarantine results should be objective and must be tested with a photometer equipped with a 450nm filter. A positive result is valid only if both the negative and positive controls have acceptable optical density readings.
Principle The detection of pro-E.coliO157:H7 is based on enzyme immunoassay (EIA) using specific monoclonal antibodies. The inner surface of the polystyrene microwell is coated with monoclonal antibody against E.coli O157:H7 antigen, and the sample and control are added into the well. If the E.coliO157:H7 antigen is present in the sample, it will be absorbed by the specific antibodies adsorbed on the wells. After washing, a binding agent is added to bind to the E.coliO157:H7 antigen absorbed by the antibody. Rinse the wells again to remove unbound binding reagent before adding enzyme substrate. When the reaction is stopped with stop solution, the blue color changes to yellow. The presence or absence of E.coliO157:H7 antigen in the sample depends on the optical density of the color produced.
(5) Automatic enzyme-linked fluorescence immunoassay system (VIDAS) screening method
The antigen identification of E.coliO157:H7 is based on enzyme-linked fluorescence immunoassay performed in the VIDAS instrument analyze.
A pipette-like device is a solid phase container (SPR), which serves as both a solid phase and a pipette during analysis. SPR is coated with a highly specific monoclonal antibody cocktail. A certain amount of enrichment broth is added to the reagent strip, and the mixture in the broth circulates inside and outside the SPR within a specific time. If E.coliO157:H7 antigen is present, it binds to the monoclonal antibody coated in the SPR, and other unbound compounds are washed away. The antibody bound to alkaline phosphatase circulates inside and outside the SPR and binds to the E.coliO157:H7 antigen bound to the inner wall of the SPR. The final flushing step flushes away the unbound conjugate. The substrate 4-methylumbelliferone is converted into a fluorescent product 4-methylumbelliferone by the enzyme on the SPR wall. Fluorescence intensity was measured by an optical scanner. The experimental results are automatically analyzed by the computer, and the test value based on the fluorescence test is compared with the standard and then a positive or negative result report for each tested sample is printed.
(6) E.coliO157:H7 rapid enzymatic reaction chromogenic medium method
The rapid enzymatic reaction is based on the fact that bacteria can synthesize and release certain substances during their growth and reproduction. For specific enzymes, select corresponding substrates and indicators according to the characteristics of the enzyme, and prepare them in the relevant culture medium. According to the obvious color change after bacterial reaction, the suspicious strain to be isolated is determined. The reaction measurement results are helpful for rapid diagnosis of bacteria. This technology organically combines traditional bacterial separation with biochemical reactions and makes the detection results intuitive, becoming an important development direction for the development of microbial detection in the future.
(7) DNA probe technology
DNA probe technology is a newly developed specific, sensitive and rapid detection method, but it has a certain proportion of false positive reactions. , so all positive results must be confirmed using standard culture methods.
Principle: Use specific DNA probes to detect E.coliO157:H7 ribosomal RNA (rRNA). The samples to be tested undergo pre-enrichment, selective enrichment and post-enrichment to dissolve the bacteria. Add labeled E.coliO157:H7 opposite-sex DNA probe for liquid phase hybridization. If there is rRNA of E.coliO157:H7 in the sample to be tested, the fluorescein-labeled detection probe and the polydeoxyadenylicacid (polydA) end capture probe will hybridize with the target rRNA sequence. Then insert the probe coated with polydeoxyadenylicacid (polydT) (solid phase) into the hybridization solution. Base pairing between PolydA and poldT facilitates probe capture: the target hybrid nucleic acid molecule is bound to the solid support, and unbound probes are washed away. Stems were incubated in horseradish peroxidase-antifluorescein conjugate. The adapter binds to the fluorescein label present on the hybridization detection probe, and unbound agent is washed away. And the measuring rod is cultured in the enzyme substrate-chromogen solution. Horseradish peroxidase reacts with the enzyme substrate to convert the chromogen into a blue compound. Once it encounters acid, the reaction stops and the color of the chromogen changes to yellow. The absorption value is measured at 450nm. If the absorption value is greater than the critical value, it indicates that E.coliO157:H7 is present in the sample to be tested.
(8) Polymerase Chain Reaction (PCR) Technology
The principle of PCR technology is to improve the sensitivity of DNA probes. The target DNA sequence can be amplified first to increase the amount of DNA. To achieve sufficient detection volume, if the reaction is based on kinetics, it can be quantitatively analyzed. In 1983, Millus and Cetus invented the most basic method of amplifying DNA or increasing the number of special nucleotide fragments in a sample - the polymerase chain reaction, or PCR method. The PCR method is based on a three-step repeated reaction. ① Denature and cleave double-stranded DNA into single-stranded DNA through heat treatment; ② Anneal and extend the primer to the specific oligonucleotide; ③ Enzymatic extension of the primer and DNA pairing to synthesize the template, primer annealing, denatured DNA fragments, and primer hybridization to form a template Can participate in another reaction. Nucleotides in the solution are polymerized by enzymes into complementary DNA fragments, and can be re-cleaved into single-stranded DNA to serve as a template for the next PCR replication. Therefore, the specific DNA will be doubled in each cycle. Typical amplification can cause 1 million-fold amplification after 20 to 40 cycles. Introducing Taq polymerase into the PCR reaction enables semi-automated and simple reaction procedures.
PCR reactions using amplified DNA have unparalleled advantages.