CN-116660232-B - Food-borne pathogenic bacteria detection method based on composite nano material

Abstract

The invention discloses a method for detecting food-borne pathogenic bacteria based on composite nano materials, which belongs to the technical field of food safety detection, wherein cerium-based nano materials DPA-Ce-GMP with enzyme-like activity are synthesized based on Ce (CH 3 COO) 3 ), guanosine-5' -disodium monophosphate (GMP) and 2, 6-pyridine dicarboxylic acid (DPA), prussian blue is taken as a carrier, vancomycin is used for identifying gram-positive bacteria, a composite nano material PB@DPA-Ce-GMP@Van with targeting and oxidase characteristics is constructed, PCV is abbreviated as PCV, PCV can be accurately adsorbed on the surface of the gram-positive bacteria to form a PCV/gram-positive bacteria complex, and PCV remained in supernatant after centrifugation can quench fluorescence of Scopoletlide (SC), meanwhile, fluorescent intensity of fluorescent red dye (AR) is increased, and fluorescence intensity ratio (SC/AR) is taken as detection signal output.

Inventors

• WANG TIANLIN
• HUANG XIANQING
• LI TIANGE
• Fu Yana
• SONG LIANJUN
• LIU XIAOMENG
• MA YAN
• LI NING
• QIAO MINGWU

Assignees

• 河南农业大学

Dates

Publication Date

20260512

Application Date

20230616

Claims (5)

1. 1. A method for detecting food-borne pathogenic bacteria based on composite nano materials is characterized by comprising the following steps: s1, preparing a bacterial liquid to be tested of food-borne pathogenic bacteria: The gram-positive bacteria in the freezing tube are dipped by an inoculating loop, and strain culture is carried out by a streak inoculation method, and bacterial liquid to be detected with the bacterial concentration of 0-10 7 CFU/mL is obtained after gradient dilution; s2, synthesizing a biological sensing system material PB@DPA-Ce-GMP@Van: prussian Blue (PB) with the concentration of 0.1-0.3 mg/mL is taken as a carrier, after ultrasonic dissolution, guanosine-5' -disodium monophosphate (GMP) and Ce (CH 3 COO） 3 ) are added into a mixed solution, the mixed solution is subjected to shaking and mixing uniformly by a mixer, then centrifugation is carried out, supernatant liquid is removed, deionized water is used for washing, deionized water is added for re-dissolution, 2, 6-pyridine dicarboxylic acid (DPA) is added after mixing uniformly, when the color becomes transparent blue, the solution is irradiated for 2-4 hours under ultraviolet, and then vacuum freeze drying is carried out for standby, so that PB@DPA-Ce-GMP solution is obtained; Redissolving the precipitate in deionized water, adding vancomycin (Van) with the concentration of 10-20 mug/mL, and carrying out ultrasonic treatment to finally obtain a biological sensing system material PB@DPA-Ce-GMP@Van; s3, detecting food-borne pathogenic bacteria: Adding gram-positive bacterial liquid with the bacterial concentration of 0-10 7 CFU/mL in the step S1 into the biological sensing system material PB@DPA-Ce-GMP@Van synthesized in the step S2, incubating for 2 hours at room temperature, centrifuging, taking supernatant, and incubating to form a PB@DPA-Ce-GMP@Van/gram-positive bacterial complex; The PB@DPA-Ce-GMP@Van in the supernatant quenches the fluorescence of scopoletin lactone (SC), meanwhile, the fluorescence intensity of fluorescent red dye (AR) is increased, the fluorescence intensity ratio SC/AR is used as a detection signal to be output, the fluorescence response of a sensing system is taken as an ordinate, the bacterial solution concentration of gram positive bacteria is taken as an abscissa, the detection data is processed, and then linear fitting is carried out, so that a linear regression equation is finally obtained.
2. 2. The method for detecting food-borne pathogenic bacteria based on composite nano materials according to claim 1, wherein in the step S1, the specific process of strain culture is that streaking inoculation is carried out on a nutrient agar culture medium, the culture is carried out in a 37 ℃ incubator for 24-48 h, colonies with good forms are picked up and inoculated in LB broth for shaking culture, after washing and centrifugation with PBS buffer solution, the colonies are resuspended in sterilized Tris-HCl buffer solution, 100 mu L of bacteria liquid flat plate is coated for flat plate counting, and an ultraviolet-visible spectrophotometer is used for measuring the optical density value OD of the bacteria suspension at a wavelength of 600nm to determine the corresponding bacteria concentration.
3. 3. The method for detecting food-borne pathogenic bacteria based on composite nano materials according to claim 1, wherein in the step S2, PB is synthesized by adding polyvinylpyrrolidone and potassium ferricyanide into hydrochloric acid, stirring for 0.5-1 h at room temperature, heating for 20-25 h in an oil bath pot at 80-90 ℃, changing the color of the solution from yellow to blue, cooling to 40-50 ℃ after heating, centrifuging the reaction solution, washing and precipitating with ethanol and water for multiple times, and finally vacuum drying the obtained PB, and preserving at 4 ℃ for standby.
4. 4. The method for detecting food-borne pathogenic bacteria according to claim 1, wherein in the step S2, preferably, the PB concentration is 0.1mg/mL, the ultraviolet irradiation time is 2.5h, and the vancomycin concentration is 10. Mu.g/mL.
5. 5. The method for detecting food-borne pathogenic bacteria based on composite nano materials of claim 1, wherein in the step S3, when gram-positive bacteria are selected as staphylococcus aureus S.aureus, a linear regression equation of concentration and fluorescence response of the S.aureus bacteria liquid is finally obtained, wherein y=0.2151x+0.3117 and R2=0.983, wherein R2 represents a correlation coefficient between variables, and the larger the value is, the better the fitting degree is.

Description

Food-borne pathogenic bacteria detection method based on composite nano material Technical Field The invention relates to a detection method of food-borne pathogenic bacteria, in particular to a detection method of food-borne pathogenic bacteria based on a composite nano material, and belongs to the technical field of food safety detection. Background Food-borne pathogenic bacteria are pathogenic bacteria that are food-borne and adhere to food products by forming a biofilm that survives over time on surfaces that come into contact with the food products, resulting in food contamination, reduced product quality and shelf life, and potential disease transmission. Contamination of food by food-borne pathogens (e.g., salmonella, escherichia coli, staphylococcus aureus, and vibrio parahaemolyticus) has evolved into a major public health problem, causing significant economic losses worldwide. Traditional plate counting methods with good specificity and reliability have been accepted as the standard method for current bacterial detection, but they generally require time-consuming culture and laborious isolation, as well as stringent laboratory conditions to perform. The detection method of large-scale instrument, including gas chromatography, liquid chromatography, atomic absorption spectrometry and the like, can realize high-sensitivity quantitative detection of target bacteria in a short time, but the detection result is easy to be false positive, and relatively expensive instrument and complex operation are required, so that the detection cannot be carried out on site. The detection methods can not meet the requirements of rapid detection of large-scale foods and on-site rapid detection of related foods at home and abroad. The effective detection of pathogens in food is a necessary premise for timely preventing and controlling food-borne epidemic. At present, the detection technology based on the fluorescence method has the advantages of rapidness, convenience, simplicity in operation, visualization, high sensitivity and the like, so that the detection technology has great potential in rapid detection of food safety. The advent of nanomaterials has further advanced the use of fluorescence methods, and a range of inorganic or organic composite nanomaterials have been used as fluorescent biosensors to detect bacteria. According to the invention patent with the application publication number of CN109856389A, the preparation of nano magnetic particles based on quantum dots and the application of the nano magnetic particles in the detection of multiple food-borne pathogenic bacteria are disclosed, a conjugate of functionalized nano magnetic particles and fluorescent marker molecules is combined with target pathogenic bacteria, an antibody or an aptamer of the fluorescent marker molecules and an antibody of the functionalized nano magnetic particles are combined with antigens of the target pathogenic bacteria, optical detection is carried out by using a fluorescence spectrometer, the types of pathogenic bacteria are determined by emission wavelength, and the number of pathogenic bacteria is detected by emission intensity, so that the multiple food-borne pathogenic bacteria in a food sample can be realized. However, the above-mentioned patents not only use an antibody as a recognition element, which is relatively expensive, but also have only a single signal channel to be correlated, which is easily affected by factors such as instrument parameters, micro-environments around bacteria, local concentration variation of probes, and excitation light sources, thus resulting in false positive or false negative results. Therefore, a method for detecting food-borne pathogenic bacteria based on composite nano materials is needed to solve the problems of high detection cost, complex operation, signal interference by external environment and the like. Disclosure of Invention The invention aims to solve the problems in the prior art and provide a food-borne pathogenic bacteria detection method based on a composite nanomaterial, which has the advantages of good biocompatibility, high sensitivity, strong anti-interference capability, simple detection operation process, low cost and good linear range and detection limit of detection results. In order to achieve the aim, the invention adopts the following technical scheme that the food-borne pathogenic bacteria detection method based on the composite nano material comprises the following steps: s1, preparing a bacterial liquid to be tested of food-borne pathogenic bacteria: The gram-positive bacteria in the freezing tube are dipped by an inoculating loop, and strain culture is carried out by a streak inoculation method, and bacterial liquid to be detected with the bacterial concentration of 0-10 7 CFU/mL is obtained after gradient dilution; s2, synthesizing a biological sensing system material PB@DPA-Ce-GMP@Van: PB with the concentration of 0.1-0.3 mg/mL is taken as a carrier, after