1 molecular biology methods
1. 1 nucleic acid probe method [1]
Cell nucleic acid DNA and RNA are the only macromolecules that can transmit genetic information, and they can be used as detection targets. The target is usually a specific nucleic acid sequence, which can be detected by using complement nucleic acid molecules as probes. Similar to immunological methods, probes also need to attach appropriate labels, such as radioisotopes, enzymes or luminescent labels. The probe technology used in previous research can only be used for special laboratory applications because it uses radioactive isotopes, so the second generation technology based on nucleic acid hybridization-colorimeter is popular now. This method relies on the detection of nucleic acid components stored during the development of ribosome R N A(t R N A). Using this natural target sequence rich in rRNA makes it possible to detect without radiation, while maintaining the sensitivity equivalent to or higher than that of radioisotope method. Generally speaking, nucleic acid probe technology is an ideal rapid detection technology, but there are also some problems, that is, it is necessary to prepare probes for detecting each strain, and probes for all strains have not been established yet, so this technology needs further development.
1.2 polymerase chain reaction (PCR) [1]
Polymerase chain reaction PCR (1) was invented by American scientist Mullis in 1983 as a method for rapid amplification of specific genes or DNA sequences in vitro. So it is also called gene amplification in vitro. It can establish a reaction in a test tube. After a few hours, it can amplify a very small number of target genes or specific DNA fragments by hundreds of thousands or even millions of times. That is, picogram (Pg) level DNA can be specifically amplified to a detectable microgram (G) level without going through a lengthy and time-consuming gene cloning procedure. A sufficient number of accurate DNA copies can be obtained. At present, PCR technology has been applied to microbial detection in three ways:
(1) Using a set of specific primers or a set of primers consisting of a specific primer and a universal primer, this PCR technique only produces one product.
(2) The species and subspecies of spoilage bacteria can be identified by random amplified polymerase chain reaction (RAPD-PCR) with arbitrary primers and a group of DNA fragment mixtures with different lengths.
(3) Nested polymerase chain reaction technology, because some microorganisms will interfere with PCR, researchers further developed PCR technology, which is the application of nested polymerase chain reaction technology. The mechanism of the difference between this technology and the above two methods is that two sets of primers are needed in the operation. The first group is used to generate amplified D N A fragments, which contain the binding sites of the second round of PCR primers and the products of the first round of reaction.
Transfer to the second round of P C R primers to amplify the target D N A.
2 immune detection technology
2. 1 Basic principles of immunoassay [2]
Immunology is based on the specific recognition and binding reaction of antigen and antibody. Antigen refers to a substance that can stimulate the immune system of animals to produce antibodies or sensitized lymphocytes, and can specifically bind with corresponding antibodies or sensitized lymphocytes in vivo or in vitro. As an antigenic substance, it should have the characteristics of foreign body, high molecular weight and complex molecular structure. Many macromolecules in food or food (including protein, enzymes, polysaccharides, nucleic acids and infected microorganisms, etc. ) are good antigens, and some small molecules (such as hormones, mycotoxins or pesticide residues) can be combined with macromolecular carriers (usually protein) to prepare artificial antigens. So in theory, almost all substances encountered in food science can be directly or indirectly used as antigens. Antibody is one of the products of immune response caused by antigen, which can react specifically with antigen, so that accurate qualitative and quantitative analysis can be carried out.
2.2 The main immunodetection technologies used in food inspection
Enzyme-linked immunosorbent assay [3]
The basis of ELISA is the immobilization of antigen or antibody and the enzyme labeling of antigen or antibody. Its basic principle is to combine antigen or antibody on the surface of solid carrier in advance without destroying its immune activity. During the determination, the tested sample (including the antibody or antigen to be detected) and the enzyme-labeled antigen or antibody react with the antigen or antibody bound on the solid carrier according to a certain procedure to form an antigen or antibody complex; When the reaction is terminated, the amount of enzyme-labeled antigen or antibody bound to the solid carrier (immune complex) is proportional to the amount of antibody or antigen to be detected in the sample. After washing, other substances in the reaction solution were removed. After adding the enzyme reaction substrate, the substrate is catalyzed by the enzyme on the solid support to become a colored product. Finally, the content of the substance to be detected in the sample can be determined by qualitative or quantitative analysis of the amount of colored products. Because of the high catalytic efficiency of enzyme, the result of immune reaction is indirectly amplified, which makes the determination extremely sensitive. China's research work in this field is relatively late, but some progress has been made.
(2) Monoclonal antibody detection technology
Monoclonal antibody (monoclonal antibody) with single antigen specificity was prepared by cell culture and cell fusion technology. It has stable performance, can recognize the subtle differences of antigen structure, can specifically bind with antigen, avoid the interference of other molecules, and can accurately determine the content of antigen. Polyclonal antibodies are often used as specific antibodies in immunological methods, but there are few reports of monoclonal antibodies, let alone those used for food detection. However, monoclonal antibodies have good repeatability, strong specificity and are not easy to cross-react, and their specificity has attracted the attention of scholars all over the world.
(3) Other immunoassay methods
Early radioimmunoassay (RIA) has been rarely used because of the use of radioactive markers and the lack of room for improvement. In recent years, there have been some successful reports on the detection of food by other immune methods, such as immunomagnetic bead enrichment technology; There are also successful examples of using monoclonal antibody colloidal gold method (MOP) [4], which uses highly specific antigen, antibody reaction and immunochromatographic analysis technology, and does not need
Pretreatment of samples.
3 rapid specimen method
Rapid test refers to taking paper, paper film, film, etc. As the carrier of culture medium, and attached with specific culture medium and chromogenic substance.
The method of determining microorganisms in food by their growth and color development [5]. This is a combination of chemistry, polymer and microbiology.
Comprehensive detection method. Rapid test paper has obvious advantages [6]: First, rapid test paper can determine a small number of samples without preparation.
Reagent, no need for a large number of glassware, simple and fast operation; It is easy to sterilize and store, convenient to transport and carry, low in price, and has no other waste liquid except paper, which greatly reduces or eliminates the pollution to the environment, and reduces the workload of cleaning after testing. Besides, hot Joan is avoided.
Lipid method is not suitable for the recovery of damaged bacteria, but suitable for laboratory, production site and field environment. Second, the quick test strip may
So as to be inoculated at the same time when sampling, and the result can better reflect the real number of bacteria in the sample at that time, so as to prevent the inoculation time from being prolonged due to bacterial reproduction.
The number has increased. Thirdly, conventional methods generally take a long time and require a specific temperature, which makes many grass-roots units and food enterprises impractical.
Stone, also can't achieve the purpose of timely discovery. There is no need to prepare samples before use, which greatly shortens the time.
Immunomagnetic bead method (IMS)
Immunomagnetic bead separation method can couple specific antibodies to the surface of magnetic particles. And specifically binds to the microorganism to be detected in the sample. Full of
The magnetic microspheres of pathogenic microorganisms gather in the direction of magnetic poles under the action of external magnetic field. Therefore, the target microorganism can be specifically and rapidly removed from the sample.
Break up quickly. Compared with the general bacterial culture and separation methods, this method greatly improves the detection rate of pathogenic Vibrio parahaemolyticus in food;
Compared with conventional detection methods, immune magnetic separation technology has obvious advantages. Can be quickly selected from the suspension containing a large number of miscellaneous bacteria.
Selective separation of target microorganisms. If combined with direct microscopy, enzyme-linked immunosorbent assay, PCR and other rapid detection methods, immunomagnetic separation technology can greatly improve the detection efficiency of pathogenic microorganisms [7].
5 gene chip method
Gene chip technology is to dot various gene oligonucleotides on the surface of the chip, amplify the DNA of microbial samples by PCR, and prepare fluorescent markers.
Probe, then hybridize with oligonucleotide spots on the chip, and finally quantitatively analyze the fluorescence distribution pattern through a scanner to determine whether there is anything in the detected sample.
Some specific microorganisms. Gene chip technology can realize the parallel detection of Qualcomm and microorganisms in the environment. Theoretically, it can be used in experiments.
All potential pathogens can be detected. The same chip can be used to detect various genetic indexes of the same pathogen. At the same time, it is sensitive, concrete and
It is rapid and convenient, and has a good development prospect in the detection of pathogenic microorganisms. The mixed genome microarray constructed by Borucki et al. [8], such as 1, can accurately identify various Listeria monocytogenes isolates: Vol0kh ov[9] is detected by single-tube multiplex amplification and gene chip technology.
And identified six kinds of Listeria: Call et al [10] Through the analysis of Sh ig a-like toxin I, Sh ig a-like toxin II and hemolysin A of Escherichia coli 0 157: H 7, it was found that the gene chip could accurately detect various strains of Escherichia coli 0 157: H 7. Although great progress has been made in sample collection, processing and gene chip technology. However, in order to apply gene chip technology to detect food microorganisms more widely, it is necessary to solve the problems of establishing standards.
Simplify procedures, reduce detection costs, simplify sample preparation and labeling operations, and further improve the specificity of detection.
Microorganisms in cosmetics widely exist in nature. The raw materials and additives of cosmetics contain a lot of nutrients such as oil, gum, protein and polyol, which are necessary nutrients such as carbon source, nitrogen source and water for the growth and reproduction of microorganisms. Under suitable conditions such as temperature and humidity, microorganisms will grow and multiply in cosmetics. When microorganisms multiply to a certain number, on the one hand, microorganisms absorb and decompose the effective ingredients in cosmetics, and some new substances are produced due to excretion, which destroys the ingredients of cosmetics, leads to mildew and corruption, discolors cosmetics (produces red, black and green mildew spots) and produces unpleasant smells, and some microorganisms also produce toxins, which can cause many serious hazards to consumers such as bacterial infections and allergies. Microbial contamination of cosmetics can be divided into two situations. Generally, microbial pollution in the process of cosmetics production is called primary pollution. The pollution suffered by consumers in the process of use is called secondary pollution. 1. The primary microbial pollution of cosmetics refers to the pollution in the production process of cosmetics, including the pollution of equipment, raw materials, production and packaging. Production equipment such as pumps, grinders, mixers, emulsifying machines, filling machines and other equipment are places where microorganisms gather. Therefore, disinfection, sterilization and other treatments are needed. Cosmetic raw materials are the pollution sources of cosmetics microorganisms. It may be contaminated before manufacturing, such as heating and stirring. Therefore, it is necessary to prevent the pollution of raw materials, mainly animal and plant raw materials, especially protein, starch and other raw materials before manufacturing. Before manufacturing, it is necessary to spot-check raw materials and count microbial cultures. The situation of microorganisms in raw materials is directly related to products, and the number of microorganisms in raw materials or products should be controlled below 100 /g, and there should be no pathogenic bacteria. In addition to disinfection and sterilization of raw materials, attention should also be paid to microbial pollution of water. Deionized water used in production cannot be stored. In summer, the number of microorganisms in deionized water can reach 1 10,000 /g, and tap water is often polluted by bacteria, so the water should also be treated, usually by heating and ultraviolet irradiation. In the manufacturing process of cosmetics, such as making emulsion, heating sterilization is usually used. Heat water to 90℃ for 20 minutes for sterilization, and then emulsify it with oil phase at similar temperature. Packaging, the last process of cosmetics production, is also a link that is easy to cause microbial pollution. Air purification is required in the packaging room, and the air cleanliness is ammonium per liter of air >: the average dust particle of 0.5μm indicates the grade. In recent years, the classification of air cleanliness in China has begun to adopt American standards, which are specifically expressed as: air cleanliness grade >; The average value of 0.5μm dust particles is100 (Ⅰ)