CN101609095A - A new colloidal gold immunochromatographic method and colloidal gold immunoassay test strip for rapid quantitative detection of Listeria monocytogenes - Google Patents

Abstract

The colloidal gold immunochromatography method for detecting Listeria monocytogenes established by the invention can quickly, sensitively, specifically and accurately detect Listeria monocytogenes in a sample, and can realize quantification, and is suitable for on-site rapid detection. The invention belongs to the field of biological detection, and in particular relates to a rapid qualitative and quantitative detection method for the detection of Listeria monocytogenes and a colloidal gold immunological detection test strip.

Description

A new colloidal gold immunochromatographic method and colloidal gold immunoassay test strip for rapid quantitative detection of Listeria monocytogenes

technical field

The invention belongs to the field of biological detection, and in particular relates to a rapid qualitative and quantitative detection method for the detection of Listeria monocytogenes and a colloidal gold immunological detection test strip.

Background technique

Listeria monocytogenes (Listeria monocytogenes, Listeria monocytogenes), is the most important human food-borne pathogenic bacteria in Listeria (Listeria), is also a kind of zoonotic pathogenic bacteria, belongs to Listeria It can cause meningitis, encephalitis, sepsis, endocarditis, miscarriage, stillbirth and abscess in humans and animals, and the mortality rate of the patients can reach 30%-40%. In recent years, many countries have reported food poisoning incidents caused by contamination of Listeria monocytogenes. Therefore, government departments around the world are paying more and more attention to food poisoning caused by Listeria monocytogenes, and have formulated some new food safety regulations, and included Listeria monocytogenes into the mandatory inspection items.

At present, the detection method of Listeria monocytogenes mainly relies on traditional separation and cultivation (referring to: Wu Qingping. "Research progress in detection technology of Listeria monocytogenes", J. Chinese Journal of Health Inspection, 2005, 15 (7): 888-890; Robin L T Churchill, Hung Lee, et al. "Detection of Listeria mono2 cytogenes and the toxinlisteriolysin O in food" J.Journal of Mi 2 crobiological Methods, 2006, 64: 141-170.) and biochemical identification, the method Time-consuming. Colloidal gold rapid diagnostic test strip technology is a new type of in vitro diagnostic technology developed since the 1990s (see: Li Yongqin, Yang Ruifu. "Immune colloidal gold rapid test with membrane as solid carrier", J. Microbiology Immunology Advances in Science, 2003, 31(1): 74-78.). In recent years, this method has developed rapidly and has been widely used in the field of biomedicine, especially in medical testing, but there are few products used in the field of food hygiene. In this study, an immunocolloidal gold test strip for the detection of Listeria monocytogenes in food contamination was developed for Listeria monocytogenes. The colloidal gold detection method established in this study is sensitive, accurate and specific, and can be further applied to the actual work of inspection and quarantine departments for the detection of Listeria monocytogenes in imported and exported foods. After this method is established, it can be complementary to the classical conventional method, and it is expected to have broad application prospects in inspection and quarantine, food industry departments and health monitoring departments, and have certain economic and social benefits. At the same time, it can provide a scientific basis for the revision or addition of international methods for microbiological testing of food.

Contents of the invention

Listeria monocytogenes is listed as one of the seven major food-borne fatal pathogens in the United States, and it has been listed as one of the key food-borne pathogens in the WHO food safety work plan. Ready-to-eat foods are easily contaminated with L. monocytogenes, which requires rapid testing of ready-to-eat batches in a timely manner.

The invention relates to a method for rapid and quantitative detection of Listeria monocytogenes by colloidal gold immunochromatography technology and a product thereof. The method of the present invention utilizes colloidal gold labeling and double-antibody sandwich immunochromatography technology to establish a rapid detection method for Listeria monocytogenes, evaluates its specificity and sensitivity, and performs quantitative detection by fitting a detection curve; in milk powder, milk, The simulated contaminated samples of Listeria monocytogenes were added to the ham sausage and other food samples to evaluate the detection ability of the method for solid, semi-solid, liquid and other foods and suspected bioterrorism samples. The method of the invention can complete the qualitative and semi-quantitative detection within 15 minutes, the sensitivity is 1ng/ml, the linear range is 105cfu/ml-108cfu/ml, and the recovery rate is 98%-101%. The method has good specificity and stability, and can directly detect samples. The colloidal gold immunochromatography method for detecting Listeria monocytogenes established by the invention can quickly, sensitively, specifically and accurately detect Listeria monocytogenes in a sample, and can realize quantification, and is suitable for on-site rapid detection.

A method for detecting Listeria monocytogenes, which includes mixing the sample to be tested with the sample diluent, and then adding the sample mixture to the sample hole of the test paper, the liquid in the sample moves upward by siphon action, and the result is interpreted within 10-15 minutes ; Described test paper comprises:

(1) reaction support;

(2) Absorbent pad;

(3) Nitrocellulose membrane, which is coated with detection bands and quality control bands for Listeria monocytogenes antibodies and quality control antibodies;

(4) Gold-labeled antibody pad, which contains colloidal gold-labeled Listeria monocytogenes P60 antibody;

(5) sample pad;

Among them, the Listeria monocytogenes antibody can be a polyclonal antibody or a monoclonal antibody; the quality control antibody can be selected from sheep anti-rabbit, mouse anti-rabbit, human anti-rabbit, rabbit anti-sheep, mouse anti-sheep, human anti-sheep, sheep Anti-mouse, human anti-mouse, rabbit anti-mouse IgG.

In the detection test paper described in the method of the present invention, wherein the absorbent pad is selected from filter paper, the reaction support is selected from PVC board, the material of the gold-labeled antibody pad is selected from polyester film, glass fiber or filter paper fiber, and the material of the sample pad is selected from polyester film. , glass fiber or filter paper fiber.

The test paper described in the method of the present invention, wherein water absorption pad, gold standard antibody pad, reaction support, nitrocellulose membrane and sample pad are constituted according to the mode shown in accompanying drawing 1; Reaction support 5 is positioned at bottom layer, and nitrocellulose membrane 2 is positioned at reaction. In the middle of the support 5, the T place of the membrane is the detection band coated with Listeria monocytogenes P60 polyclonal antibody, and the C place is the quality control band coated with goat anti-rabbit IgG; the glass fiber membrane 3 is located at One side of the upper part of the nitrocellulose membrane and partly overlapped with it, and the membrane contains colloidal gold-labeled Listeria monocytogenes P60 polyclonal antibody; the absorbent pad 1 is located on the other side of the upper part of the nitrocellulose membrane 2 relative to the glass fiber membrane 3 One side and overlap with 2 parts. Sample pad 4 is located on the opposite side of 1 from 2 and partially overlaps 3 .

In the test paper described in the method of the present invention, one side of the water-absorbing pad is the starting end, and the side of the glass fiber membrane is the end, the detection antibody strip is located near the end, and the quality control strip is close to the initial end.

In the test paper described in the method of the present invention, wherein the antibody against Listeria monocytogenes can be a polyclonal antibody or a monoclonal antibody; the quality control antibody can be selected from goat anti-rabbit, mouse anti-rabbit, Human anti-rabbit, rabbit anti-goat, mouse anti-goat, human anti-goat, goat anti-mouse, human anti-mouse, rabbit anti-mouse, etc. IgG.

In the test paper described in the method of the present invention, the concentration of the anti-Listeria monocytogenes P60 polyclonal antibody is 0.5-5 mg/ml. The concentration of quality control antibody is 0.1-2mg/ml. The amount of the anti-Listeria monocytogenes P60 antibody to label 1ml of colloidal gold is 5-20ug.

The preparation method of the detection test paper of Listeria monocytogenes of the present invention, the method comprises:

(1) Spray the nitrocellulose membrane with two strips of anti-Listeria monocytogenes antibody and quality control antibody with a certain spray film speed with a flow-separating spray gold marking machine;

(2) Prepare a glass fiber membrane containing a colloidal gold-labeled anti-Listeria monocytogenes antibody, uniformly coat the colloidal gold-labeled anti-Listeria monocytogenes antibody on the glass fiber membrane, and dry or freeze-dry .

The application of the test paper of the present invention in the detection of Listeria monocytogenes comprises mixing the sample to be tested with the sample diluent, and then adding the sample mixture to the sample hole of the test paper, and the liquid in the sample moves upward by siphon action, 10-15 minutes to interpret the results.

The invention also provides a test paper for detecting Listeria monocytogenes prepared by the method.

Antigen and antibody are adopted in the method of the present invention, wherein Listeria monocytogenes is such as Listeria monocytogenes, bacterium number 19111, can be purchased from the Institute for the Control of Pharmaceutical and Biological Products of the Ministry of Health; Listeria monocytogenes P60 polyantibody is such as Listeria monocytogenes, Multiple cloning antibody of P60 can be purchased from Jilin Provincial Inspection and Quarantine Bureau; goat anti-rabbit IgG can be purchased from Dingguo Biotechnology Company. Here, the polyclonal antibody or monoclonal antibody that binds to the antigen can be used, but the P60 polyclonal antibody used in this experiment is from the Jilin Bureau; and the detected object here is a bacterial fluid, not an antigen, but the antigen is used Based on the principle of specific antibody binding, the detected object can be other Listeria monocytogenes strains. This experiment uses one of the Listeria monocytogenes strains in our Institute of Inspection and Quarantine, and the bacterial number is 19111.

Chromatography test strip consumables are used in the product and method of the present invention, such as binding pad (glass fiber), nitrocellulose membrane (NC membrane, SHF 1350225), sample pad and absorbent pad, filter paper, purchased from Minipore company.

According to the detection test paper, the absorbent pad is made of filter paper, the reaction support is made of PVC board, the material of the gold-labeled antibody protective film is selected from polyester film, glass fiber or filter paper fiber, and the material of the sample pad is selected from polyester film, glass fiber or filter paper fiber.

The experimental instrument used in the present invention is, for example, a gold standard immunoassay analyzer, which can be purchased from Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences and jointly developed by China Academy of Inspection and Quarantine.

Description of drawings

Figure 1 shows the detection sensitivity of Listeria monocytogenes colloidal gold immunochromatographic test strips.

Figure 2 is a schematic diagram of specific detection results; among them, 1: Listeria monocytogenes, 2: Staphylococcus aureus, 3: Escherichia coli O157:H7, 4: Botulinum toxin, 5: Yersinia pestis, 6: Abbrin toxin 7: blank control.

Figure 3 is the fitting curve of the colloidal gold immunochromatographic test strip detection system for Listeria monocytogenes; X: the T/C ratio of the reading value of the gold standard immunoassay analyzer at each concentration; Y: the concentration of Listeria monocytogenes.

Figure 4 shows the stability test of colloidal gold immunochromatographic test strips for Listeria monocytogenes; among them, 1: Listeria monocytogenes 10 ⁸ CFU/mL, 2: 1% BSA, 3: Staphylococcus aureus 10 ⁸ CFU /mL, 4: blank control.

Detailed ways

Embodiment 1: the preparation of colloidal gold immunochromatography test strip

1. Antigen and antibody

Listeria monocytogenes is Listeria monocytogenes, bacteria number 19111, from the Institute for the Control of Pharmaceutical and Biological Products of the Ministry of Health,

Listeria monocytogenes P60 polyantibody is Listeria monocytogenes, multiplecloning antibody of P60 comes from Jilin Inspection and Quarantine Bureau),

Goat anti-rabbit IgG can be purchased from Dingguo Biotechnology Company.

2. Consumables for chromatography test strips

Bonding pad (glass fiber), nitrocellulose membrane (NC membrane, SHF 1350225), sample pad, absorbent pad, and filter paper were purchased from Minipore.

3. Experimental equipment

Gold Standard Immunity Analyzer (jointly developed by Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences, and China Academy of Inspection and Quarantine).

4. Preparation of colloidal gold immunochromatographic test strips

4.1 Colloidal gold bonding pad

The colloidal gold particles of 25 nm were prepared by labeling the Listeria monocytogenes P60 polyclonal antibody with a pH value of 7.5 to 8.0 and a concentration of 0.2 mg/ml in the sodium citrate method, and dried at 37° C.

4.2 Nitrocellulose membrane

Detection band: Listeria monocytogenes antibody P60 polyclonal antibody 1.5 mg/ml+2% BSA; quality control band: goat anti-rabbit IgG: 1 mg/ml, dry at 37°C [6-9].

4.3 Assembly

Paste the sample pad, bonding pad, and water-absorbing pad on the backing card with the release agent in sequence, cut into 0.4cm strips, dry, pack in a shell, and store at room temperature for later use.

Embodiment 2: Qualitative and quantitative detection of samples

1. Sample processing

Use the sample treatment solution (5mM PB pH7.4) to carry out gradient dilution of viscous liquid samples such as ham sausage, milk powder, milk, etc., add 1/100 (W/V) to the sample treatment solution to dissolve the solid food such as ham sausage, and take it at rest. Clear, stand still and take the supernatant. Different doses of Listeria monocytogenes were added as mock test samples.

2. Qualitative detection

Add 100ul of the treated sample and sample treatment solution (as a negative sample) to the end of the prepared chromatographic strip sample pad, let it stand for 15min, and observe the result. If both the test strip and the quality control strip appear red, it is judged as positive, and if only the quality control strip appears red, it is negative.

3. Quantitative detection

3.1. Determination of judgment value

According to 1.6.1, test the negative samples 20 times, scan and read the signal T/C ratio with a gold standard immunoassay analyzer. The sum of the average (AVERAGE) and 3 times the standard deviation (STDEVA) of the T/C ratios of 20 samples is the CUT-OFF value.

3.2. Quantitative detection and judgment

Put the color-developed gold-labeled strip into the gold-labeled immunoassay analyzer for scanning, and read the signal T/C ratio, if it is greater than the judgment value, it is positive.

Embodiment 3: sensitivity test

1. Sensitivity of quantitative detection

Detecting different concentrations of Listeria monocytogenes, the calculated judgment value (CUT-OFF) is 0.017, and the average T/C ratio of Listeria monocytogenes with a concentration of 10 ¹ cfu/ml and 10 ² cfu/ml is the average value. 0.00, lower than 0.017, it is negative; the average T/C ratio of Listeria monocytogenes from 10 ³ cfu/ml to 10 ⁸ cfu/ml is higher than 0.017, the result is positive; when Listeria monocytogenes When the bacterial concentration is greater than 10 ³ cfu/ml, the average T/C ratio is greater than 0.017, so the quantitative detection sensitivity is 10 ³ cfu/ml.

Table 1 Detection results of various concentrations of Listeria monocytogenes colloidal gold immunochromatographic test strips

2. Intuitive detection sensitivity

Colloidal gold immunochromatographic test strips were prepared according to the determined optimal reaction conditions to detect different concentrations of Listeria monocytogenes. Dilute Listeria monocytogenes with sample treatment solution to a concentration of 10 ⁸ cfu/ml; 10 ⁷ cfu/ml; 10 ⁶ cfu/ml; 10 ⁵ cfu/ml; 10 ⁴ cfu/ml; 10 ³ cfu/ml ; 10 ² cfu/ml; 10 ¹ cfu/ml, to be tested at the same time. The results are shown in Figure 1. The sensitivity is 10 ⁴ cfu/ml and the color development is clearer.

Embodiment 4: standard curve fitting experiment

Take T/C as the abscissa, and take LOG10 sample concentration values (10 ¹ cfu/ml, 10 ² cfu/ml, 10 ³ cfu/ml, 10 ⁴ cfu/ml, 10 ⁵ cfu/ml, 10 ⁶ cfu/ml, 10 ⁷ cfu/ml, 10 ⁸ cfu/ml) is the ordinate, fitting the standard curve (see Figure 3).

For each concentration of Listeria monocytogenes detected, the judgment value (CUTOFF) is the T/C reading value of 0.017. It can be seen that less than 10 ³ cfu/ml is negative, and the linear relationship between 10 ⁵ cfu/ml-10 ⁸ cfu/ml is good, and the fitting curve is shown in Figure 3. The linear equation is: y=99.701x-9.502; R ² =0.997.

Embodiment 5: specificity test

Prepare colloidal gold immunochromatographic test strips according to the determined optimal reaction conditions, and use the sample diluent to treat Staphylococcus aureus, Escherichia coli O157:H7, botulinum toxin, Yersinia pestis and other cocci or bacilli separately to 1×108CFU /ml, detected with the prepared colloidal gold immunochromatographic test strip, and compared with the sample of Listeria monocytogenes with the same number of bacteria, and detected the blank control at the same time. The result is shown in Figure 2.

Embodiment 6: recovery rate test

Within the linear detection range, detect the known concentration of Listeria monocytogenes, calculate the detection concentration according to the T/C ratio read by the gold standard analyzer and the standard curve, and the ratio percentage of the detection concentration to the theoretical concentration is the recovery rate. It can be seen from Table 2 that the recovery rate is calculated according to the fitting curve equation with LOG10 sample concentration (10 ¹ cfu/ml-10 ⁸ cfu/ml) and T/C value, and it can be seen that the recovery rate is between 10 ⁵ cfu/ml-10 ⁸ cfu/ml The recovery rate is between 98%-101%.

Table 2 Recovery rate of Listeria monocytogenes colloidal gold immunochromatography test strip detection system

Embodiment 7: stability test

Take the Listeria monocytogenes colloidal gold test strip placed at 37°C for detection. The test results on the 7th day show that the Listeria monocytogenes test strip has good stability and can still be specific after being placed at 37°C for 7 days. Listeria monocytogenes was detected, and the sensitivity did not decrease. Compared with the newly prepared test strip, the sensitivity did not decrease significantly, and the specificity was good. See Figure 4, in which: 1. Listeria monocytogenes 10 ⁸ CFU/mL, 2. 1% BSA, 3. Staphylococcus aureus 10 ⁸ CFU/mL, 4. Blank control; Listeria monocytogenes colloidal gold test strips can still detect Listeria monocytogenes 108CFU/mL, and there is no cross-reaction with other food-borne bacteria, and the stability is good.

Embodiment 8: the experiment of detection sample

Add 50mg of ham sausage, 50mg of milk powder, and 250μl of milk to 1ml of the sample diluent containing Listeria monocytogenes, mix well, let stand for 10min, and then take 1 part of the cultured bacteria solution, and use PB10 times serial dilution for detection . The test results of the test strip detecting simulated contamination samples show that the test strip can still detect 105 CFU/ml of simulated contaminated Listeria monocytogenes in food.

in conclusion:

The invention is a double-antibody sandwich colloidal gold immunochromatographic test strip suitable for on-site rapid detection. The label of the test strip and the labeled antibody are combined through electrostatic attraction and hydrophobic interaction, so it will not affect the activity of the antibody. Colloidal gold itself has a color visible to the naked eye, and the results can be interpreted without an instrument. There is no need for washing, and the labeled antibodies that do not form immune complexes are automatically separated by chromatography, which not only simplifies the operation steps, but also reduces the interference factors that affect the experimental results.

The method of the present invention can usually complete the detection in 5-10 minutes, the sample processing method is simple, flexible in operation, convenient in transportation, does not need other auxiliary equipment, and the result can be judged intuitively, and the test strip has its own quality control line, and the experimental results are clear at a glance. On-site detection provides the best detection method, especially for the detection of ready-to-eat food contaminated by Listeria monocytogenes. It has the characteristics of simplicity, rapidity, specificity, sensitivity, and stability. Therefore, the test strip has broad application prospects.

In this study, the colloidal gold test strip and the colloidal gold biosensor were organically integrated into a colloidal gold quantitative detection system. The advantage of using the colloidal gold immunoassay analyzer is that the reader can correctly interpret the test result of the test strip when the human eye cannot correctly identify whether the suspicious substance is positive or not. The error rate caused by human subjective interpretation is reduced, and the objectivity and accuracy are increased; and. And through the optimization of the entire detection system, the phenomenon of false positives in the detection of serum, food, beverage and other samples can be overcome, and quantitative detection can be realized. In addition, the colloidal gold interpreter is easy to carry, easy to use, accurate in judgment, objective in results, and easy to keep, which brings convenience to the inspection and testing work.

Claims (9)

1, a kind of method that detects Listeria monocytogenes comprising with sample to be measured and sample diluting liquid mixing, adds sample mix liquid test paper sample well place again, and the liquid in the sample relies on syphonic effect up, 10-15 minute sentence read result; Described test paper comprises:

(1) reaction holder;

(2) adsorptive pads;

(3) nitrocellulose membrane, this film are coated with Listeria monocytogenes antibody and Quality Control detection of antibodies band and Quality Control band;

(4) golden labeling antibody pad wherein contains the Listeria monocytogenes P60 antibody of colloid gold label;

(5) sample pad;

Wherein, Listeria monocytogenes antibody can be polyclonal antibody, also monoclonal antibody; Quality Control antibody can be selected from the IgG of goat-anti rabbit, mouse-anti rabbit, the anti-rabbit of people, the anti-sheep of rabbit, mouse-anti sheep, the anti-sheep of people, sheep anti mouse, the anti-mouse of people, the anti-mouse of rabbit.

2, method according to claim 1, wherein, adsorptive pads is selected filter paper for use, and the reaction holder is selected the PVC plate for use, and the material of golden labeling antibody pad is selected from polyester film, glass fibre or filter paper fibre, and the material of sample pad is selected from polyester film, glass fibre or filter paper fibre.

3, method according to claim 1 and 2, wherein, reaction holder (5) in the described test paper is positioned at bottom, nitrocellulose membrane (2) is positioned at the middle part on the reaction holder (5), the T place of this film is the test strip of Listeria monocytogenes P60 polyclonal antibody bag quilt, and the C place is the Quality Control band of goat anti-rabbit igg bag quilt; Gold labeling antibody pad (3) is positioned at a side on nitrocellulose membrane top and overlaps with it, and this pad contains the Listeria monocytogenes P60 polyclonal antibody of colloid gold label; Adsorptive pads (1) is positioned at the opposite side for golden labeling antibody pad (3) on nitrocellulose membrane (2) top and overlaps with nitrocellulose membrane (2), and sample pad (4) is positioned at upward opposite with adsorptive pads (1) side of nitrocellulose membrane (2) and overlaps with golden labeling antibody pad (3).

4, method according to claim 3, wherein, adsorptive pads one side is an initiating terminal, and sample pad one side is terminal, and the band that detects antibody is positioned near terminal, and the Quality Control band approaches initiating terminal.

5, method according to claim 3, wherein, the antibody of described Listeria monocytogenes is Listeria monocytogenes P60 polyclonal antibody, its concentration is 0.5-5mg/ml.

6, method according to claim 5, wherein, the Quality Control antibody concentration is 0.1-2mg/ml.

7, method according to claim 6, wherein, the amount of described anti-Listeria monocytogenes P60 antibody labeling 1ml collaurum is 5-20ug.

8, the preparation method of the detection test paper of the described Listeria monocytogenes in each described method of a kind of claim 1-7, this method comprises:

(1) uses the nitrocellulose membrane that sprays anti-Listeria monocytogenes antibody and two bands of Quality Control antibody every stream metal spraying pen machine with certain spray film speed;

(2) the golden labeling antibody pad that is made of glass fibre membrane of a kind of anti-Listeria monocytogenes antibody that contains colloid gold label of preparation is uniformly coated on the anti-Listeria monocytogenes antibody of colloid gold label on the glass fibre membrane, and oven dry or freeze drying.

9, by the test paper of the detection Listeria monocytogenes of the described method of claim 8 preparation.

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