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At present, the rapid detection methods of Staphylococcus aureus can be roughly divided into three types: one is the rapid detection medium method; the second is the immunological method; the third is the nucleic acid-based molecular biology method [1].
China Microbial Species Inquiry Network
1 rapid detection medium method
1.1 chromogenic medium detection
Chromogenic/Fluorogenic Culture Media (Chromogenic/Fluorogenic Culture Media) is a new type of medium for detecting microorganisms by using the principle of the color reaction of the enzymes produced by the microorganisms and the corresponding chromogenic substrate to reduce the pure culture and further biochemistry of the strains. Identification step [2].
CHROMagar Staph aureus (CSA), developed by Dr. Alain in 1974, is a major marker enzyme of Staphylococcus aureus DNase and coagulase, with toluidine blue, methyl green, acridine orange and 5-bromo-4-chloro -3å²å“š-thymidine-3 phosphate or the like is a chromogenic substrate to identify a chromogenic medium of the bacterium. The Baird Parker+Rabbit Plasma Fibrinogen (RPF) medium developed by Mérieux, France, is a gray-to-black and opaque colony that grows as a Staphylococcus aureus. Because the medium contains rabbit plasma, it is not necessary. The plasma coagulase test was further confirmed [3]. Huang Jicheng et al [4] used this medium to compare with the national standard method, the total coincidence rate was 98%, and the detection time was greatly shortened. In addition, China's Qingdao Haibo Biotech Co., Ltd. has also developed similar products, which are now widely used.
1.2 Paper method
The presence of the bacteria is detected by reacting a thermostable nuclease produced by S. aureus with a developer to form a pink ring.
PetrifilmTM Staphylococcus aureus test piece produced by 3M Company of the United States. The test piece is composed of two parts, one part is modified Baird-Parker medium, which has strong selectivity to Staphylococcus aureus, and the other part contains color development. Reagent and deoxyribonucleic acid (DNA) reaction sheet. Schoeller [5] compared the traditional method of S. aureus in food with the 3M paper method. The test proved that the 3M paper has strong specificity and short detection time. Wu Zhongliang et al [6] used this test piece for food samples, and the results showed that the detection rate of PetrifilmTM was 93.3%.
2 immunological detection methods
The use of antigen and specific antibody specific binding on the theoretical basis for the detection of Staphylococcus aureus, simple operation, strong specificity, high sensitivity, suitable for the detection of large quantities of samples.
2.1 Enzyme-Linked Immunosorbent Assay (ELISA)
The ELISA method is the most commonly used method in immunodiagnosis, and the "sandwich method" is often used in the detection of Staphylococcus aureus. Schotte [7] has reported a modified ELISA method, a rapid immunochromatography manual method, that can detect 500 pg/mL of Staphylococcus aureus enterotoxin in 15 min. However, the ELISA method also has some defects. The reagents used in the test are highly selective, expensive, and susceptible to environmental, temperature, and time conditions.
2.2 Immunofluorescence Assay (IFA) technology
According to the reaction characteristics of antigen-antibody specific binding, a known antibody and a fluorescent dye are chemically combined to form a fluorescent label, and under certain conditions, the sample is subjected to a binding reaction thereto, and then a fluorescent label is observed under a fluorescence microscope. The antigen-antibody complex is present and, if present, the sample is shown to contain S. aureus. In 1991, Rowe et al [8] achieved the purpose of detecting high molecular weight Staphylococcus aureus enterotoxin and low molecular weight Staphylococcus aureus enterotoxin in mixed samples by immunofluorescence. In 2004, Tim Alefantis [9] developed a method for rapid detection of Staphylococcus aureus enterotoxin based on immuno-double antibody sandwich fluorescence method. Compared with the traditional method, the detection time is greatly shortened, and the whole process takes 40 to 50 minutes.
2.3 Reverse Passive Latex Agglutination (RPLA)
The RPLA method uses the principle of indirect agglutination reaction to adsorb specific antibodies on the latex particles. When the antigen and antibody specifically bind, the latex undergoes agglutination reaction, and the degree of aggregation is proportional to the bacterial content in the sample to be tested. The Slidex Staph-kit from Mérieux France can read the results in 20 s [10]. Trisum's Aureus TsetTM can observe results within 1 min [11]. Although RPLA is simple and easy to perform, the sensitized latex is prone to self-coagulation, which affects the sensitivity of the reaction, resulting in inaccurate detection results. In addition, the method can only detect enterotoxin of Staphylococcus aureus.
3 molecular biology testing
3.1 Polymerase Chain Reaction (Polymerase Chain Reaction, PCR)
This method overcomes the shortcomings of the traditional detection methods, and has been developed rapidly in recent years and has been applied in various fields. The earliest foreign report using PCR to detect Staphylococcus aureus from food was reported in 1991. WILSON et al. [12] used this method to detect Staphylococcus aureus enterotoxin in artificially contaminated dried skim milk within 8 hours. B and C1, target DNA detection limit reached 1 fg (3.3 gene chip technology
Gene chip technology is a high-tech of reverse solid phase hybridization which has been rapidly developed in recent years. It has the characteristics of high throughput, rapidity and sensitivity. The principle is to design an oligonucleotide for detecting Staphylococcus aureus. The needle is fixed on the nitrate membrane in a certain manner to prepare a gene chip, and the target gene is amplified and labeled by the primer, and hybridized with the gene chip under appropriate conditions, and the presence or absence of Staphylococcus aureus is identified according to the hybridization result. Li Xiuping et al [15] have used this technology to detect the main pathogens of cow mastitis, with good specificity, high accuracy and operability, which provides a powerful basis for clinical treatment.
3.4 Loop-mediated isothermal amplification (LAMP)
The technique designed four specific primers for six regions of the target gene, and reacted under isothermal conditions by strand displacement DNA polymerase. The nucleic acid amplification reaction was completed within 1 h, and the LAMP characteristic ladder-like strips were exhibited. The amplification result can be judged by visual observation. It does not require thermal denaturation of the template, long-term temperature cycling, and cumbersome electrophoresis. It also does not require expensive equipment, is fast and sensitive, has high specificity, and low cost, and is especially suitable for detection of large, immediate, and on-site pathogenic microorganisms. It is a truly popular nucleic acid detection method and is easy to promote at the grassroots level. In 2003, it was gradually used for clinical clinical testing. In 2005, it was applied in the detection of animal diseases. Wu Shaoqiang et al. [16] established the RT-LAMP detection method for Asian type I foot-and-mouth disease virus, using agarose gel electrophoresis and color change. The results of the determination of precipitation and other phenomena provide a simpler and faster method for on-site rapid detection of foot-and-mouth disease, meeting the needs of on-site quarantine. Yang Qiulin et al [17] used LAMP technology to detect Toxoplasma gondii, showing good specificity and sensitivity. Jiang Yanzeng et al [18] used the LAMP method to detect African swine fever virus, which is 100 times more sensitive than the OIE standard PCR method and has no cross-reactivity with other common porcine DNA viruses. With the continuous improvement of this technology, it is believed that LAMP technology will fully play its role in various fields.
Research progress on rapid detection methods of Staphylococcus aureus
Staphylococcus aureus Gram-positive cocci, widely distributed in air, water, soil, feed, but also in the body surface, nose, throat and intestine of humans and animals, belonging to zoonotic pathogens. Among them, Staphylococcus aureus has the strongest pathogenicity. In addition to causing inflammation of skin tissues and organs, the resulting toxins contaminate food and cause food poisoning. In recent years, there have been many food poisoning incidents caused by Staphylococcus aureus. According to the US Centers for Disease Control and Prevention, food poisoning caused by Staphylococcus aureus ranks second, second only to E. coli, accounting for 33% of all bacterial food poisoning cases, and Canada is as high as 45%. At the same time, Staphylococcus aureus is also one of the main causative factors causing mastitis in dairy cows, causing huge economic losses to the breeding industry.