Rapid detection method for florfenicol in meat matrix

Abstract

The invention provides a rapid detection method for florfenicol in a meat matrix. The rapid detection method comprises the steps of sample treatment, magnetic nano material enrichment, washing and elution and the like. According to the rapid detection method for florfenicol in the meat matrix, the meat is pretreated, florfenicol in the meat matrix is fully extracted, then the florfenicol is enriched through a magnetic nano material and finally removed to serve as a detection solution, and then detection is conducted through a rapid detection card, so that the sensitivity of rapid detection is fully improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a detection method, in particular to a rapid detection method of florfenicol in meat matrix. BACKGROUND

[0002] Florfenicol is an antibiotic drug, which is widely used in the feeding process of cattle, sheep, chicken and the like. Florfenicol has a broad-spectrum bacteriostatic effect by inhibiting the activity of peptidyl transferase, and has a broad antibacterial spectrum, including various gram-positive, gram-negative bacteria and mycoplasma and the like. In order to avoid the florfenicol residue in meat, it is necessary to detect whether the meat contains florfenicol. At present, the determination of florfenicol and florfenicol amine residues in animal food according to the national food safety standard adopts liquid chromatography-tandem mass spectrometry method. The liquid chromatography-tandem mass spectrometry method has high detection precision, but the liquid chromatography mass spectrometry equipment is expensive, and some small farms or laboratories are difficult to afford the equipment cost. Therefore, the test paper is usually used to roughly detect the florfenicol residue in meat.

[0003] At present, there are many florfenicol test papers on the market. Among them, the colloidal gold florfenicol test paper is widely used in the rapid screening process of meat due to the advantages of simple operation, low cost, and the result can be observed and judged by naked eye. However, the sensitivity of the colloidal gold florfenicol test paper is relatively low.

[0004] In order to solve the above problems, people have been seeking an ideal technical solution. SUMMARY

[0005] The present application aims at the deficiencies of the prior art, and provides a rapid detection method of florfenicol in meat matrix.

[0006] In order to achieve the above object, the technical scheme adopted by the present application is:

[0007] A rapid detection method of florfenicol in meat matrix, comprising the following steps:

[0008] S1. symmetrical sample is subjected to crushing treatment, placed in a centrifugal tube, vortex mixed, and then added with anhydrous methanol, oscillated, and then centrifuged and separated, and the supernatant is reserved;

[0009] S2. The magnetic nanometer material is placed in a centrifugal tube, anhydrous methanol is added, mixed, and then centrifuged and separated, and the supernatant is removed; then purified water is added, mixed, and then centrifuged and separated again, and the supernatant is removed;

[0010] S3. The supernatant reserved in step S1 and the magnetic nanometer material treated in step S2 are mixed and oscillated, centrifuged and separated, and the supernatant is removed;

[0011] S4. Washing the magnetic nanomaterial treated in step S3;

[0012] S5. Eluting the magnetic nanomaterial treated in step S4, and testing the eluent.

[0013] In step S1, 1.5-3.5 ml of anhydrous methanol is mixed with 1 g of the sample and oscillated for 5-15 min.

[0014] In step S3, 3-8 mg of the magnetic nanomaterial corresponds to 1 ml of the supernatant.

[0015] In step S2, 0.1-0.2 ml of anhydrous methanol corresponds to 1 mg of the magnetic nanomaterial.

[0016] In step S4, the magnetic nanomaterial is washed with purified water for 1-2 times, the purified water is mixed with the magnetic nanomaterial uniformly, and then centrifugal separation is performed, the supernatant is removed, and the process is recorded as one washing process.

[0017] In step S5, 20%-40% of methanol is first mixed with the magnetic nanomaterial treated in step S4, and then purified water is added and mixed uniformly, centrifugal separation is performed, and the supernatant is the eluent.

[0018] The particle size of the magnetic nanomaterial is 15-50 microns.

[0019] The present application has outstanding substantial characteristics and significant progress compared with the prior art. Specifically, the present application is a rapid detection method for florfenicol in meat matrix, which comprises the steps of sample treatment, magnetic nanomaterial enrichment, washing and elution. The florfenicol in the meat matrix is fully extracted through pretreatment of the meat, and then enriched by the magnetic nanomaterial, and finally eluted as a detection solution, and then detected by a rapid detection card, thereby fully improving the sensitivity of rapid detection. Specifically, in the sample extraction step, anhydrous methanol is used to denature the protein, so that the florfenicol is fully separated from the protein and is easy to extract; then the florfenicol is adsorbed and enriched by the hlb solid-phase extraction magnetic beads, and then washed to avoid the influence of impurities, and finally eluted to obtain the detection solution. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a comparison chart for detecting the test solution prepared according to example 1 and comparative examples 3-4 from a 30 ug / kg chicken sample.

[0021] Figure 2 is a comparison chart for detecting the test solution prepared according to comparative example 2 from a 30 ug / kg, 40 ug / kg, 50 ug / kg, and 60 ug / kg chicken sample. DETAILED DESCRIPTION

[0022] The technical solutions of the present application are described in further detail below through specific embodiments. In the following examples, the florfenicol detection uses the florfenicol rapid detection card of Wuxing Biological Technology Co., Ltd. (the detection limit of the tissue sample is 100 ug / kg), and the magnetic nanomaterial uses product number 71113-1 hlb solid-phase extraction magnetic beads, the sample uses chicken breast meat, removes the fat fascia, and the chicken breast meat is detected by an instrument method to determine that it does not contain florfenicol and its similar drugs, thereby obtaining a blank sample. Specifically, the chicken breast meat is derived from 100-day-old Shaobu chickens, which are pre-fed for 20 days before the test, fed with full-price feed without adding any drugs, and free water is provided. After 20 days, the chickens are slaughtered, and the chicken breast meat is taken. In addition, the blank sample and florfenicol are used to prepare 5 ug / kg, 10 ug / kg, 20 ug / kg, 30 ug / kg, 40 ug / kg, 50 ug / kg, 60 ug / kg, 80 ug / kg, 100 ug / kg, and 120 ug / kg chicken samples, respectively.

[0023] Example 1

[0024] The present embodiment provides a rapid detection method for florfenicol in meat matrix, which comprises the following steps:

[0025] S1. First, the sample is extracted. 0.5 g of minced chicken breast meat sample (accurate to 0.01 g) is weighed and placed in a centrifuge tube, vortexed for 30 s, and allowed to stand for 1 min. 1.5 ml of anhydrous methanol is added. Shake for 10 min, then centrifuge at 10000 r / min for 5 min, and take the supernatant for standby;

[0026] S2. Take 5 mg of hlb solid-phase extraction magnetic beads in a new 2 ml centrifuge tube, add about 0.5 ml of anhydrous methanol to activate, mix well for 10 s by ultrasonic or vortex shaker, then place the centrifuge tube in a magnetic separator and centrifuge until the solution is clear. Remove the supernatant with a pipette. Add 0.5 ml of purified water, mix well for 10 s, then place the centrifuge tube on the magnetic separator and centrifuge until the solution is clear. Remove the supernatant with a pipette;

[0027] S3. Add 1 ml of the supernatant obtained in step S1 to the hlb solid-phase extraction magnetic beads treated in step S2, vortex shake at the maximum speed of the vortex shaker for 1 min to fully combine the analyte and the magnetic beads, then place the centrifuge tube in a magnetic separator until the solution is clear. Remove the supernatant with a pipette;

[0028] S4. Add 0.5 ml purified water to the magnetic beads after step S3 processing, mix well with vortex oscillator for 30 s, then place the centrifuge tube on the magnetic separator until the solution is clear, and remove the supernatant with a pipette; add 0.5 ml purified water to the magnetic beads again for the second time, mix well with vortex oscillator for 30 s, then place the centrifuge tube on the magnetic separator until the solution is clear, and remove the supernatant with a pipette;

[0029] S5. Add 75 ul 30% methanol to the magnetic beads after step S4 processing, elute the magnetic beads with vortex oscillator for 60 s, then add 175 ul purified water and mix well for 10 s, place the centrifuge tube on the magnetic separator until the solution is clear, and collect the supernatant as the test solution.

[0030] Comparative Example 1

[0031] This comparative example provides a rapid detection method for florfenicol in a meat matrix, which uses the extractant and tissue sample processing method provided by the florfenicol rapid detection card manufacturer to process the sample, as follows:

[0032] Homogenize the fat-free tissue sample, and weigh 1 g of the uniformly crushed sample into a 15 mL centrifuge tube.

[0033] Accurately add 5 ml of the extractant to the centrifuge tube, tightly seal it with a bottle plug, and shake vigorously for 2 minutes, then centrifuge at 4000 rpm for 3 minutes.

[0034] Take 0.1 mL of the supernatant (do not suck up the oil) into a 5 mL centrifuge tube, add 1 mL of the extractant, and mix well by hand to obtain the test solution.

[0035] Comparative Example 2

[0036] This comparative example provides a rapid detection method for florfenicol in a meat matrix, which is basically the same as Example 1, except that in step S1, anhydrous methanol is replaced by 30% methanol.

[0037] Comparative Example 3

[0038] This comparative example provides a rapid detection method for florfenicol in a meat matrix, which is basically the same as Example 1, except that in step S4, the number of washes is 1.

[0039] Comparative Example 4

[0040] This comparative example provides a rapid detection method for florfenicol in a meat matrix, which is basically the same as Example 1, except that step S4 is removed and no washing is performed.

[0041] The detection conditions of Example 1 and the comparative examples are shown in Table 1.

[0042] Table 1

[0043] Chicken test sample Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 10 ug / kg Negative / Negative Negative Negative 20 ug / kg Positive / Negative Positive Negative 30 ug / kg Positive / Negative Positive Positive 40 ug / kg / / Negative / / 50 ug / kg / / Positive / / 60 ug / kg / / Positive / / 80 ug / kg / Negative / / / 100 ug / kg / Positive / / / 120 ug / kg / Positive / / /

[0044] The test sample of 30 ug / kg chicken was prepared according to Example 1, Comparative Examples 3-4 to prepare the test solution for comparison, and the test card detection result was positive. The typical picture is shown in Figure 1 .

[0045] The test sample of 30 ug / kg, 40 ug / kg, 50 ug / kg, 60 ug / kg chicken was prepared according to Comparative Example 2 to prepare the test solution, and the test sample of 30 ug / kg chicken was prepared according to Example 1 to prepare the test solution for comparison. The test card detection result is shown in Figure 2 , the test sample of 30 ug / kg, 40 ug / kg chicken was negative, and the test sample of 50 ug / kg, 60 ug / kg chicken was positive, which indicated that the sample treated with anhydrous methanol can promote protein denaturation, fluorophenical dissolution, and improve the detection sensitivity.

[0046] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: the specific embodiments of the present application can be modified or some technical features can be replaced by equivalent; without departing from the spirit of the technical scheme of the present application, it should be covered in the technical scheme range of the present application claimed by the present application.

Claims

1. A rapid detection method for florfenicol in meat matrix, comprising the following steps: S1. Take symmetrical samples, crush them, place them in centrifuge tubes, vortex and mix, let stand, then add anhydrous methanol and shake, then centrifuge and separate, and keep the supernatant for later use; S2. Place the magnetic nanomaterial in a centrifuge tube, add anhydrous methanol, mix well, then centrifuge and remove the supernatant; then add purified water, mix well, centrifuge again and remove the supernatant. S3. Mix the supernatant obtained in step S1 with the magnetic nanomaterials processed in step S2, oscillate, centrifuge, and remove the supernatant; S4. Wash the magnetic nanomaterials processed in step S3. S5. The magnetic nanomaterials treated in step S4 are eluted, and the eluent is used for testing.

2. The rapid detection method for florfenicol in meat matrix according to claim 1, characterized in that: In step S1, each gram of sample is mixed with 1.5-3.5 ml of anhydrous methanol and shaken for 5-15 min.

3. The rapid detection method for florfenicol in meat matrix according to claim 1, characterized in that: In step S3, each milliliter of supernatant corresponds to 3-8 mg of magnetic nanomaterials.

4. The rapid detection method for florfenicol in meat matrix according to claim 1, characterized in that: In step S2, each milligram of magnetic nanomaterial corresponds to 0.1-0.2 ml of anhydrous methanol.

5. The rapid detection method for florfenicol in meat matrix according to claim 1, characterized in that: In step S4, the material is washed 1-2 times with purified water. The purified water and magnetic nanomaterials are mixed evenly, then centrifuged to remove the supernatant. This is recorded as one washing process.

6. The rapid detection method for florfenicol in meat matrix according to claim 1, characterized in that: In step S5, 20%-40% methanol is first added and mixed with the magnetic nanomaterials treated in step S4, then purified water is added and mixed well, centrifuged, and the supernatant is the eluent.

7. The rapid detection method for florfenicol in meat matrix according to claim 1, characterized in that: The particle size of the magnetic nanomaterials is 15-50 micrometers.

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