Colloidal gold immunochromatography detection method for tetrodotoxin in blood and kit thereof

Abstract

The invention relates to a colloidal gold immunochromatography detection method for tetrodotoxin in blood and a kit thereof.The method comprises the following steps that 1, an extracting agent is added into a blood sample, vortex oscillation and centrifugation are conducted, supernate is taken, the extracting agent is a mixed solution of acetic acid and acetonitrile, and the extracting agent is a mixed solution of ethyl acetate and acetonitrile; the adding amount of the extracting agent is 390-410 [mu] L per 200 [mu] L of blood sample; 2, carrying out nitrogen blowing drying on the supernate obtained in the step 1; 3, a resolvent is added into the residues obtained after nitrogen blowing drying, a solution to be detected is obtained after redissolving, the resolvent is a PBS buffer solution, and the adding amount of the resolvent is 145-160 microliters. According to the method, key parameters of the blood sample pretreatment step are optimized, blood matrix interference is effectively removed, the colloidal gold immunochromatography process is ensured to be smoothly carried out, and therefore rapid, sensitive and reliable qualitative screening of tetrodotoxin in blood is achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a colloidal gold immunochromatographic detection method for tetrodotoxin in blood and a kit thereof. BACKGROUND

[0002] Tetrodotoxin is a potent neurotoxin commonly found in puffer fish and other organisms. Mis-eating can cause severe poisoning and even death. In the forensic identification of poisoning cases, clinical first aid and food safety monitoring, it is crucial to quickly and accurately detect tetrodotoxin in biological samples such as blood and urine.

[0003] Currently, the detection methods for tetrodotoxin in biological samples mainly include biological detection, liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), enzyme-linked immunosorbent assay (ELISA), and biosensor method. However, these methods have certain limitations: biological detection has large individual differences and poor specificity; instrument analysis (such as UPLC-MS / MS) requires expensive and precise instruments, complex sample pretreatment procedures, and professional operators, and is time-consuming, making it difficult to meet the demand for on-site rapid screening; ELISA method has relatively complicated operation steps and takes a long time; and the biosensor method has problems such as poor signal stability and reproducibility.

[0004] In contrast, colloidal gold immunochromatographic technology has the outstanding advantages of simple and quick operation, visual results, strong specificity, low cost, and no need for complex instruments, and is very suitable for on-site rapid screening and large-scale preliminary screening. Currently, there are colloidal gold rapid detection products for tetrodotoxin in aquatic product matrices on the market. However, blood samples are complex and contain a large amount of interfering substances such as proteins, lipids, and hemoglobin, which can seriously interfere with the smooth progress of colloidal gold immunochromatographic detection, specifically: masking the normal color development of the detection line (T line) and the quality control line (C line), hindering the chromatography movement of the test liquid on the nitrocellulose membrane, and causing a significant decrease in detection sensitivity or even detection failure. Directly using the existing pretreatment methods for aquatic products or urine for blood detection cannot obtain effective detection results. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a colloidal gold immunochromatographic detection method for tetrodotoxin in blood and a kit thereof. The method optimizes the key parameters of the blood sample pretreatment step, effectively removes the interference of the blood matrix, ensures the smooth progress of the colloidal gold immunochromatographic process, and realizes the rapid, sensitive, and reliable qualitative screening of tetrodotoxin in blood.

[0006] The technical solution of the present application is to provide a colloidal gold immunochromatographic detection method for tetrodotoxin in blood, comprising the following steps,

[0007] Step 1, adding an extraction agent to the blood sample, vortexing and centrifuging, taking the supernatant, the extraction agent is a mixed solution of acetic acid and acetonitrile, the amount of the extraction agent added is 390-410 μL per 200 μL of blood sample;

[0008] Step 2, nitrogen blowing drying of the supernatant obtained in step 1;

[0009] Step 3, adding a redissolving agent to the residue after nitrogen blowing drying, obtaining a to-be-tested solution after redissolving, the redissolving agent is a PBS buffer, the amount of the redissolving agent added is 145-160 μL;

[0010] Step 4, detecting the to-be-tested solution using a colloidal gold immunochromatographic detection device, the detection device includes a detection line containing anti-tetrodotoxin specific antibodies.

[0011] As preferred, the following steps are included:

[0012] Sample extraction: 200 μL of blood sample is measured, 400 μL of extraction agent is added, the extraction agent is an acetonitrile solution containing 0.3% (v / v) acetic acid. Vortex for 1-2 minutes to fully mix and homogenize the sample, then centrifuge at a speed of about 1000 r / min for 2 minutes, and carefully transfer all the supernatant to another clean centrifuge tube.

[0013] Concentrated drying: the supernatant obtained in step 1 is placed in a 50-65°C water bath, and nitrogen (or clean air) is used to dry it, obtaining a dried extract residue.

[0014] Redissolving: 150 μL of redissolving agent is added to the above dried residue, vortexed thoroughly to completely dissolve it, and the obtained solution is the to-be-tested solution. The redissolving agent is preferably a phosphate buffer (PBS).

[0015] Detection: 100 μL of the to-be-tested solution is taken and added to the sample hole (or gold label micropore) of the colloidal gold immunochromatographic detection device, and the reaction is carried out according to the device instructions (usually after incubation for a certain time, the test strip is inserted or directly chromatographed). The detection device is a conventional competitive colloidal gold test strip or detection card, which contains a detection line (T line) coated with anti-tetrodotoxin specific antibodies and a control line (C line) coated with a secondary antibody.

[0016] Result interpretation: after the reaction is completed (usually 10-15 minutes), observe the color development of the T line and the C line on the test strip. If the C line develops color and the T line does not develop color or develops color significantly lighter than the C line, it is judged to be positive, indicating that the concentration of tetrodotoxin in the sample is higher than the detection limit of the method; if both the C line and the T line develop color and the T line develops color to a depth comparable to or deeper than the C line, it is judged to be negative; if the C line does not develop color, it indicates that the detection is invalid.

[0017] As preferred, in step 1, the concentration of acetic acid in the extraction agent is 0.3% (v / v).

[0018] As preferred, in step 1, the vortex oscillation time is 1-2 minutes, and the centrifugal speed is 1000 r / min, and the centrifugal time is 2 minutes.

[0019] As preferred, in step 2, the nitrogen blowing drying is carried out under the condition of 50-65℃ water bath.

[0020] As preferred, in step 4, the colloidal gold immunochromatographic detection device is a test strip or a detection card.

[0021] The present application also provides a kit for detecting tetrodotoxin in a blood sample, which comprises components for implementing the above method, at least including:

[0022] The extraction agent is an acetonitrile solution containing 0.3% (v / v) acetic acid;

[0023] The reconstitution agent is a phosphate buffer (PBS);

[0024] The colloidal gold immunochromatographic detection device comprises a test strip or a detection card containing a detection line containing anti-tetrodotoxin specific antibodies.

[0025] As preferred, the concentration of acetic acid in the extraction agent is 0.3% (v / v).

[0026] Further, the kit further comprises a centrifugal tube for containing the blood sample and a pipette for sucking the blood sample.

[0027] Compared with the prior art, the present application has the following advantages:

[0028] Strong specificity and good anti-interference ability: the present application can efficiently extract tetrodotoxin from blood by optimizing the extraction agent and its amount, while effectively precipitating and removing a large amount of interfering substances such as proteins and hemoglobin in blood, overcoming the direct interference of blood matrix on colloidal gold immunochromatography.

[0029] High sensitivity: by optimizing the reconstitution volume, while ensuring that there is enough sample for detection, the method realizes the moderate concentration of the target, so that the detection limit of tetrodotoxin in blood reaches 37.5 μg / L (actual application can be specified as 40 μg / L), which can meet the rapid screening demand of blood samples in poisoning cases.

[0030] The operation is quick and simple, and is suitable for on-site application: the whole pretreatment process has few steps, and the time is short (about 20 minutes can be completed), and no complex and expensive instrument is needed. The optimized nitrogen blowing time (10-12 minutes) is reasonable, the detection step is the same as the operation of the conventional colloidal gold test strip, and non-professionals can operate after simple training, and it is very suitable for large-scale rapid screening in primary laboratories, first-aid sites or law enforcement sites.

[0031] The cost is low, and the result is intuitive: based on the mature colloidal gold immunochromatography platform, the reagent cost is low, the detection result can be visually observed, and no instrument is needed for judgment.

[0032] Support for subsequent confirmation analysis: the method can be used as an efficient preliminary screening method, and the positive samples screened out can be further sent to the laboratory for confirmation and quantitative analysis by UPLC-MS / MS and other methods, which greatly improves the detection efficiency and resource utilization. DETAILED DESCRIPTION

[0033] The application will be further described in the specific embodiments:

[0034] A colloidal gold immunochromatographic detection method for tetrodotoxin in blood, comprising the following steps,

[0035] 1. Extraction of tetrodotoxin in blood

[0036] 200 μL (accurate to 0.1 μL) of mixed plasma sample was taken into a 1.5 mL polypropylene centrifuge tube, 400 μL of acetic acid solution (0.3%) - acetonitrile solution was added, vortex oscillation was performed for 1-2 min until the tissue was uniformly dispersed, 1000 r / min centrifugation was performed for 2 min, the supernatant was all transferred into a 1.5 mL polypropylene centrifuge tube, and nitrogen (air) blowing was performed under a 50-65℃ water bath, 150 μL of dilution buffer solution was added for dilution, and the solution was a test solution for colloidal gold immunochromatographic rapid detection card determination.

[0037] 2. Blood dilution

[0038] The detection limit of the kit is 20 μg / L according to the qualitative detection limit of tetrodotoxin in urine. First, refer to the extraction method of tetrodotoxin in urine, take 200 μL of blood stock solution, add 8, 16, 24, 32, 40 μL of tetrodotoxin standard solution respectively, and dilute with PBS buffer solution by 2, 4, 6, 8, 10 times respectively, so that the toxin content in the diluted blood is 20 μg / L (as shown in Table 1). Because blood contains hemoglobin, high concentration can cover the normal color of T line and C line, and affect the movement of the blood to be tested on the cellulose acetate membrane. When the dilution multiple is 2-6 times, the detection card cannot normally color, when the dilution multiple is 8 times, the detection card appears T line (light) and C (dark) line, the detection card is effective, at this time the detection limit of tetrodotoxin in blood is 160 μg / L, which is much higher than the detection limit in urine, and has no practical application value, so this method cannot be used.

[0039] Table 1 blood dilution gradient and corresponding tetrodotoxin concentration

[0040]

[0041] 3. Blood extraction agent dosage

[0042] Tetrodotoxin is a small molecule polar substance, and a suitable purification method needs to be selected to reduce the interference of such matrix. According to the related literature report, 0.3% acetic acid + acetonitrile is used as the extraction agent, and the addition amount is determined by experiment. 400 uL and 600 uL of extraction agent are added to 200 μL of positive blood sample (tetrodotoxin content 37.5 μg / L) respectively, and the subsequent steps are according to 1. The experimental results show that both detection cards show positive, and the T line of 600 uL extraction agent is deeper than that of 400 uL, that is, the color of T line and C line is closer. In the nitrogen blowing step, the nitrogen blowing time of 400 uL extraction agent is 10-12 min, and the nitrogen blowing time of 600 uL extraction agent is 14-16 min. Through the nitrogen blowing time and the indication of the detection card, the addition of 400 uL can shorten the nitrogen blowing time and make the indication result clearer.

[0043] 4. Blood resolvent addition amount

[0044] After adding 400 uL of 0.3% acetic acid + acetonitrile extraction agent to the positive sample, centrifuging and nitrogen blowing, 50, 100, 150 uL of PBS buffer solution is added to the centrifuge tube for dilution, and the subsequent steps are operated. When the resolvent volume is 150 μL, the blood can move on the cellulose acetate membrane, and the T line and C line can normally color, and the detection card is effective. Generally, at least 100 μL of test liquid needs to be sucked into the micropore to react with the antibody in the hole to color, and a small amount of test liquid will be lost in the pipette suction process. To ensure that the detection card can normally color, at least 150 μL of resolvent is added to the centrifuge tube for dilution.

[0045] 5. The detection limit of tetrodotoxin in blood

[0046] Take 200 μL of blood from a healthy volunteer (male), and add 5 μL, 7.5 μL and 10 μL of 1 μg / mL tetrodotoxin standard solution, respectively, as shown in Table 2. After extraction with the extraction agent and nitrogen blowing, add 150 mL of PBS diluent, which is the test solution. The subsequent steps are operated normally.

[0047] Table 2 Standard solution volume of tetrodotoxin added to blood

[0048]

[0049] The results show that when the tetrodotoxin standard solution is added at 5 ng, the test card shows negative, and as the added amount increases, the C line is darker than the T line at 7.5 ng, indicating positive. Therefore, the detection limit of the colloidal gold immunochromatographic method for tetrodotoxin in blood after the above pretreatment optimization is 37.5 μg / L. Considering random errors and systematic errors and other factors, the detection limit of this method can be specified as 40 μg / L.

[0050] The kit for detecting tetrodotoxin in blood samples comprises components for implementing the above method, including:

[0051] The extraction agent is an acetonitrile solution containing 0.3% (v / v) acetic acid;

[0052] The reconstitution agent is a phosphate buffer (PBS);

[0053] The colloidal gold immunochromatographic detection device comprises a test strip or test card containing a detection line containing anti-tetrodotoxin specific antibodies;

[0054] and a centrifuge tube for containing the blood sample and a pipette for aspirating the blood sample.

[0055] In another embodiment, a kit comprising an optimized specification of extraction agent (0.3% acetic acid-acetonitrile, 400 μL each), reconstitution agent (PBS, 150 μL each) and commercially available colloidal gold test card is used to screen blood samples from a batch of suspected poisoning cases.

[0056] The operation process is as described in the summary. Within 15 minutes, all samples were rapidly detected, and 2 positive samples were screened out. The results are consistent with the subsequent LC-MS / MS confirmation results, proving the reliability and practicality of the method and kit of the present application.

[0057] The application successfully applies colloidal gold immunochromatography technology to detection of tetrodotoxin in blood by optimizing the pretreatment conditions of blood samples, in particular, determining the key parameters of the extraction agent (0.3% acetic acid-acetonitrile, 400 μL) and the reconstitution agent (PBS, 150 μL), establishing a rapid, sensitive, specific and simple operation screening method, and providing a corresponding kit. The method effectively solves the problem of blood matrix interference, fills the gap of on-site rapid detection technology in the field, and has important significance for public safety event emergency handling, clinical first aid and forensic identification.

[0058] The above only describes the preferred embodiments of the application, but should not be understood as limiting the claims. Any equivalent flow transformation made by using the application description is included in the patent protection scope of the application.

Claims

1. A colloidal gold immunochromatographic method for detecting tetrodotoxin in blood, characterized in that: Includes the following steps, Step 1: Add the extractant to the blood sample, vortex and centrifuge, and take the supernatant. The extractant is a mixed solution of acetic acid and acetonitrile. The amount of extractant added is 390-410 μL per 200 μL blood sample. Step 2: Dry the supernatant obtained in Step 1 by nitrogen blowing; Step 3: Add a reconstitution solvent to the residue after nitrogen blowing and reconstitute to obtain the test solution. The reconstitution solvent is PBS buffer, and the amount of reconstitution solvent added is 145-160 μL. Step 4: The test solution is tested using a colloidal gold immunochromatographic assay device, which includes a detection line containing a specific antibody against tetrodotoxin.

2. The method for colloidal gold immunochromatographic detection of tetrodotoxin in blood according to claim 1, characterized in that: In step 1, the amount of the extractant added is 400 μL per 200 μL blood sample; in step 3, the amount of the reconstitution solvent added is 150 μL.

3. The method for colloidal gold immunochromatographic detection of tetrodotoxin in blood according to claim 1, characterized in that: In step 1, the concentration of acetic acid in the extractant is 0.3% (v / v).

4. The method for colloidal gold immunochromatographic detection of tetrodotoxin in blood according to claim 3, characterized in that: In step 1, the vortex oscillation time is 1 to 2 minutes, the centrifugation speed is 1000 r / min, and the centrifugation time is 2 minutes.

5. The method for colloidal gold immunochromatographic detection of tetrodotoxin in blood according to claim 1, characterized in that: In step 2, the nitrogen blowing drying is carried out under water bath conditions of 50-65°C.

6. The method for colloidal gold immunochromatographic detection of tetrodotoxin in blood according to claim 1, characterized in that: In step 4, the colloidal gold immunochromatographic detection device is a test strip or a test card.

7. A kit for detecting tetrodotoxin, characterized in that, The kit comprises components for performing the method of any one of claims 1 to 5, including at least: The extractant is a mixed solution of acetic acid and acetonitrile; The reconstitution solvent is PBS buffer. The colloidal gold immunochromatographic assay device includes a detection line containing a specific antibody against tetrodotoxin.

8. The kit for detecting tetrodotoxin according to claim 7, characterized in that: The concentration of acetic acid in the extractant is 0.3% (v / v).

9. The kit for detecting tetrodotoxin according to claim 7, characterized in that: The kit also includes centrifuge tubes for holding blood samples and pipettes for drawing blood samples.

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