At present, feed safety issues have become an important issue of global concern, and harmful microbial contamination has become a major control indicator in feed safety. The harmful microorganisms in the feed mill mainly include harmful bacteria, molds, coliforms, etc., which are very harmful to animal and animal-derived foods. Feed production companies generally have harmful substances in the production process, including cross-contamination of harmful microorganisms. To control feed ingredients and harmful microorganisms in products, it is necessary to first have accurate and rapid methods for detecting harmful microorganisms. Classical standard detection methods are often time consuming, so rapid detection of harmful microorganisms has become a research hotspot in recent years. Based on the published research literatures at home and abroad in recent years, this paper summarizes the research progress of rapid detection methods for harmful microorganisms for reference. 1 Research progress on rapid detection methods of harmful microorganisms 1.1 Biosensor Technology A biosensor is an instrument that is sensitive to biological substances and can convert its concentration into electrical signals for detection. Biosensors are further divided into: enzyme biosensors, microbial sensors, and immunosensors. They have the advantages of low cost, small size and high sensitivity; they can obtain real-time, rapid and diversified analytical test results from corrupt or semi-corrupt foods and feeds. Zhang Jie et al. used the biotin-avidin system to link the molecular motor with the probe to construct the F0F1-ATPase molecular motor biosensor, which can specifically recognize Vibrio parahaemolyticus with a minimum detection limit of 1pg/reaction system. The rapid diagnosis of its pathogenicity eliminates the need for thermal cycling and shortens the detection cycle. The entire analysis process takes only 1h to 2 hours, and does not require complicated operations such as enzymatic digestion and electrophoresis. Zhang Jie et al. ligated a specific Salmonella-specific gene (invA as its target gene fragment) nucleic acid probe to the ε subunit of F0F1-ATPase through biotin-avidin system to construct a biosensor; After combining with the biosensor, the negative control was compared with the amount of ATP produced by catalyzing the synthesis of adenosine triphosphate (ATP) for 30 min, and the Salmonella DNA in the sample was detected. The detection time of the method for Salmonella DNA was 1 h, and the detection limit was 10 ng/ml. The results of PCR detection using 30 strains of bacteria isolated from the analysis samples were consistent with the results of PCR detection. Kim et al. designed an IMC module (immuno-magnetic concentration) linked to a real-time analysis system in order to improve the sensor's ability to analyze and detect Listeria monocytogenes. Biosensor technology greatly reduces inspection time and detects pathogenic microorganisms in hours or even minutes, providing an effective tool for rapid detection of microorganisms. However, this method is subject to certain restrictions on pretreatment. 1.2 Gene chip technology Gene chip technology is a new food-borne detection technology that is rapidly developing in the life sciences. The method combines microelectronic lithography technology with DNA chemical synthesis technology, greatly improves the probe density of the gene chip, and reduces the amount of reagents, which can realize standardization and batch mass production, and has great development potential. Combine gene chip technology with other technologies to achieve better results. Ge Heze et al. designed specific probes and primers for each bacterium according to bioinformatics technology using bacterial 16SrDNA and 23SrDNA sequences. Based on the preparation of gene chips, by amplification and hybridization of target genes and analysis of hybridization results, The detection and identification effects of common intestinal pathogenic bacteria were evaluated, and bacterial culture samples of 100 diarrhea patients were collected. In 100 clinical stool samples, 50 (50%) of intestinal pathogens were detected by gene chip technology; 47 (47%) of intestinal pathogens were identified by bacterial culture, of which 42 gene chips were included. The results were consistent with the bacterial culture results, and the accuracy rate was 79.25%. There was no significant difference between the two groups (P>0.05). The method has high throughput, high accuracy and high sensitivity, short detection time, and can simultaneously detect a variety of harmful microorganisms, and can be used as an important method for clinically identifying bacteria. It will be widely used in food and feed safety control, sudden food safety incident detection, entry and exit inspection and quarantine, etc., but the testing cost is expensive. 1.3 polymerase chain reaction technology Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments with the advantages of high specificity, high sensitivity, simplicity and speed, and low purity requirements for specimens. large. In the increasingly severe form of food safety, many scientists and technicians try to use a variety of detection methods to achieve the purpose of solving the above problems. A combination of multiple assays is designed to compensate for the limitations of a single assay. At present, there are hotspots in the international research, such as multiplex fluorescence real-time PCR detection technology, which combines the advantages of multiplex PCR and real-time PCR to monitor PCR products. Shi Jianling et al. designed primers and probes based on the 23SrDNA sequence of Helicobacter pylori, constructed plasmid standards, established a real-time PCR system and evaluated the method. The 100 clinical samples collected in 2012-05-10 were tested and applied simultaneously. The comparative analysis of immunohistochemistry and silver staining method showed that the detection range was wide, and there was a good linear relationship in 107copies/l-102copies/l (how many copies of DNA/RNA in 1l solution), and related The coefficient is 1; there is no cross-reaction with various bacteria, the specificity is good; the detection time is short, the whole PCR reaction process only takes 70min; and the measurement can be repeated in the standard batch of different copy number, and the other clinical digestive tract pathogens are not A specific amplification curve appears; real-time PCR has the advantages of high sensitivity and specificity, and good repeatability. There are also reports on the use of enzyme-linked immunosorbent assay and PCR technology to establish a method for detecting harmful microorganisms, which effectively improves the detection rate of harmful microorganisms. 1.4 Method The principle of the bioimpedance method is to rapidly detect the content of microorganisms in a sample by utilizing changes in the electrical properties of the medium caused by the metabolism of the microorganism. The impedance method has the advantage of distinguishing between living cells and dead cells, while dead cells are often not pathogenic. The key to ensuring food and feed safety is to test the number of living cells. Chen Jiang uses the impedance method to detect harmful lactic acid bacteria in beer. 630A medium (Austria SY-LAB) is more suitable for impedance method. E value is used as the criterion for the change of impedance value. Compared with the traditional medium method, the impedance method The test results are accurate and reliable, and the detection time can be shortened to 72h. This method is fast, but only determines the degree of contamination. 1.5 Protein Chip Technology Protein chip technology, also known as protein (micro) array, refers to immobilization of a large number of known proteins (such as enzymes, antigens, antibodies, receptors, ligands, cytokines, etc.) on chemically modified solid phase carriers (glass sheets, On the silicon wafer, etc., on the basis of retaining the physicochemical properties of the protein, the protein is combined with the surface of the carrier, and then the laser scanning system or the inductive coupling device is used to acquire the relevant image; then the image is analyzed by a special computer software, and the qualitative and quantitative results are analyzed. Howell et al. used a microcontact printing (CP), commonly known as soft etching, to produce an E. coli E. The antibody microarray of coli O157:H7 and renibacterium salmoninarum showed that the cross-reaction of this chip with other harmful microorganisms was less, the detection concentration was 7×107 cfu/ml, and the detection time was 40 min. Effective microbiological testing methods. Fang Zhen established the test E. Liquid phase protein chip method for coli O157:H7. The actual beef sample was detected by plotting the detection curve and applying the method and the liquid phase chip method using H2O as a solvent. Comparing the positive detection rate of the method with the bacterial isolation culture method, the lowest positive concentration can be detected as 103 cfu/ml (after sputum), and the minimum detection limit is 100 cfu/ml; there is no cross reaction with 18 intestinal bacteria. Good specificity; 3 repeated experiments, inter-assay coefficient of variation 7.61, good repeatability; using this method to detect artificially added bacteria in beef samples, the lowest concentration that can be detected positive is 0.5cfu/ml (before enrichment); Methods 154 samples of actual beef were detected and 19 positive samples were detected, while 16 samples of positive samples were detected by bacterial culture. Protein chip technology is a kind of analysis technology with high specificity, high sensitivity, high throughput, good repeatability, strong applicability, wide application range and miniaturization. It has good development prospects in various research fields of life sciences, but Like the gene chip technology, the cost of testing is high and the development is not mature. 1.6 Nano Gold Marking Technology Nanogold labeling technology refers to tiny particles with a diameter of 1 nm to 100 nm. It has high electron density, dielectric properties and catalysis. It can bind to many biomacromolecules without affecting the biological activity of biomacromolecules. In recent years, rapid detection studies based on nanogold labels have been applied in many harmful microorganisms, and there are many types of microorganisms detected. Domestic and foreign scientists and technicians have dealt with the surface characteristics of nano gold particles and specified on the surface of nano gold particles. The oligonucleotide probe was developed to develop a new microbial detection system. Liu Xia used the amino coupling method to immobilize polyclonal antibody (PAb) on the sensor surface as a primary antibody, and gold nanoparticles (AuNPs) labeled E. coli E. A polyclonal antibody (PAb) of coli O157:H7 was used as a secondary antibody by a sandwich method using a two-channel surface plasmon resonance (SPR) sensor for E. Coli O157:H7 was tested. The detection limit of SPR direct method was 103 cfu/ml, the linear range was 103 cfu/ml - 109 cfu/ml; the detection limit of gold nanoparticle enhanced sandwich method was 10 cfu/ml, and the linear range was 10 cfu/ml - 1010 cfu/ml. The sensitivity is improved by 100 times, the detection range is wider, the detection time is shortened, and the selectivity and reproducibility are better. The technology has the advantages of simplicity, rapidity, high sensitivity, high specificity, low cost, and low sample requirement. It is suitable for on-site screening and has wide application value and development prospects in feed detection. 2 Rapid detection equipment for harmful microorganisms At present, experts in the field of biology have developed a series of techniques through in-depth research on rapid detection of microorganisms, including ATP fluorometer, multi-functional microbial fluorescence detector, microbial real-time photoelectric detection system, and paper method. These rapid detection technologies for harmful microorganisms in foods can also be applied in feeds in the future. The application of these methods will provide a strong guarantee for feed safety and promote the sustainable and healthy development of China's feed industry. The following highlights the two commonly used rapid detection equipment for harmful microorganisms. 2.1 RVLM type microbial rapid detection system The high-performance portable microbiological tester produced by Royal Biotech of Germany combines the advantages of various test methods, such as culture dish method, enzymatic method (β-glucuronidase analysis), immunology (antigen search), gene The method (gene search) has many advantages such as convenience, simple operation, rapid test, high sensitivity, high specificity, low cost, qualitative and semi-quantitative measurement. 2.1.1 Rapid test object Total viable bacteria, coliform, Escherichia coli, Enterobacteriaceae, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Listeria, Enterococcus, Clostridium perfringens, Clostridium sulfite reduction, Mold (Aspergillus fungus, Aspergillus), yeast, Legionella, etc., can be quantitatively detected. 2.1.2 Rapid detection of product features Wide range of applications: It can be used in many fields such as food, feed, medicine, water quality, air microbial detection, etc. It can detect solid, liquid, gaseous and paste, slurry and other samples. Easy to use: a portable system can complete the sampling, experiment, post-processing, etc., without the need for multiple testing instruments. Simple operation: only 1ml or 1g is required for the test sample, and no pre-treatment is required, and the operation can be completed in only 3 steps. High sensitivity: Target microorganisms of the order of 1 cfu can be detected to meet domestic and international microbiological testing standards. High specificity: Specificity up to 99.999%, eliminating experimental interference caused by non-target microorganisms. High accuracy: 100% quantitative analysis. Number of inspections: 8 slots can provide 8 different experiments at the same time, 13 different standard culture environments. Quick detection: The test result can be obtained in 10 minutes at the earliest, which can be used for on-site inspection. Highly intelligent: Automatically controls incubation temperature and incubation time, and automatically generates experimental reports. Fully automated: Independently and continuously detect and record the number of microbes (cfu) in the bottle. In European laboratories, 80% - 90% of the microbiological testing experiments use the RVLM type microbial detection system, its ultra-high sensitivity can meet EU and domestic testing standards. RVLM microbial rapid detection is currently the industry's highest intelligent microbial detector. 2.2 ChemScan RDI microbial rapid detector ChemScan AES Chemunex has combined the laser scanning synthetic imaging technology with the live cell luciferase label to develop the ChemScan RDI microbial rapid detector. The instrument can quickly count microorganisms and protozoa in filterable samples within 1.5 hours, suitable for the food, feed, cosmetics and pharmaceutical industries. Its characteristics: only detect the number of living cells, the laser automatically reads the whole filter after scanning, the speed is very fast, no need to increase bacteria, the result can be obtained within 90min - 100min; optional fluorescence microscope is connected with the instrument, automatic positioning Suspected microorganisms were confirmed for observation. Sensitivity: 1 microorganism or protozoan can be detected; detection range: 1 - 5000 cells.
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