A method for detecting the bacterial quorum sensing signal molecule AI-2

Abstract

This invention provides a method for detecting the bacterial quorum sensing signal molecule AI-2, belonging to the field of biodetection technology. The method includes the following steps: extracting the sample by mixing it with hydrochloric acid aqueous solution, centrifuging the extract, and collecting the supernatant. The sample includes intestinal contents and / or rumen contents. The supernatant is reacted with sulfamic acid solution, followed by the addition of 2,3-diaminonaphthalene solution for derivatization. The derivatized product is then centrifuged, and the supernatant is collected for detection by high-performance liquid chromatography (HPLC). This method is simple, highly operable, and achieves a detection limit of 0.006 μg / mL and a quantitation limit of 0.020 μg / mL for AI-2. The relative standard deviations of intra-day precision and inter-day repeatability for retention time and peak area are both less than 5.0%, and the average recovery rate is between 65% and 101%, enabling accurate quantitative determination of AI-2.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and in particular relates to a method for detecting the bacterial quorum sensing signal molecule AI-2. Background Technology

[0002] AI-2, produced in both Gram-positive and Gram-negative bacteria, is a crucial signaling molecule for bacterial quorum sensing. AI-2 controls various bacterial functions, including bioluminescence in Vibrio harveyi and biofilm formation in many bacteria. AI-2 is produced by the LuxS enzyme, which induces the synthesis of its precursor molecule, 4,5-dihydroxy-2,3-pentanedione (DPD). The DPD form is unstable and spontaneously cyclizes to form AI-2, which exists in multiple forms within the organism. Therefore, AI-2 detection primarily focuses on detecting DPD. The gut microbiota is a vital component of the gut ecosystem, and its stability depends on quorum sensing. Under normal physiological conditions, quorum sensing may contribute to the spatial distribution of gut bacteria. Studies have shown that AI-2 is widely present in the rumen of ruminants and is closely related to nutrient metabolism; AI-2 may also be involved in the colonization of gut microbiota in livestock. Therefore, detecting AI-2 levels in intestinal contents is significant for elucidating its mechanism of action in the gut. However, current research on the detection of AI-2 molecules mainly focuses on bacterial cultures and nitrogen-fixing sludge, with almost no reports on its detection in the intestinal or rumen contents of livestock. Therefore, a method for detecting AI-2 in the intestinal or rumen contents of livestock is urgently needed. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a method for detecting the bacterial quorum sensing signal molecule AI-2, which has good accuracy and high sensitivity in detecting AI-2 in intestinal contents or rumen contents.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a method for detecting the bacterial quorum sensing signal molecule AI-2, comprising the following steps:

[0006] The sample to be extracted is mixed with hydrochloric acid aqueous solution for extraction. After obtaining the extract, the supernatant is collected by centrifugation. The sample to be extracted includes intestinal contents and / or rumen contents.

[0007] After reacting the supernatant with aminosulfonic acid solution, 2,3-diaminonaphthalene solution was added for derivatization. The derivatized product was then centrifuged, and the supernatant was collected for detection by high performance liquid chromatography.

[0008] Preferably, the mass-to-volume ratio of the sample to be extracted to the hydrochloric acid aqueous solution is 1:15 to 1:50 g / mL; and the concentration of the hydrochloric acid aqueous solution is 0.05 to 0.15 mol / L.

[0009] Preferably, the extraction is performed 1 to 2 times.

[0010] Preferably, the volume ratio of the supernatant: aminosulfonic acid solution: 2,3-diaminonaphthalene solution is 2:1:2; the concentration of the aminosulfonic acid solution is 70-90 g / L; and the concentration of the 2,3-diaminonaphthalene solution is 150-300 μg / mL.

[0011] Preferably, the reaction is carried out at room temperature for 5 to 15 minutes.

[0012] Preferably, the derivatization temperature is 85–95°C, and the derivatization time is 25–35 min.

[0013] Preferably, the intestinal contents and / or rumen contents are livestock intestinal contents and / or livestock rumen contents.

[0014] Preferably, the chromatographic conditions for high-performance liquid chromatography (HPLC) detection are as follows: the chromatographic column is a Zorbax SB-C. 18 The column temperature was 30℃, and the injection volume was 20 μL. The mobile phase consisted of mobile phase A (0.1% phosphoric acid aqueous solution) and mobile phase B (acetonitrile), with a flow rate of 0.8 mL / min. The gradient elution program was as follows: 0–4 min, mobile phase A 70%, mobile phase B 30%; 4–15 min, mobile phase A decreased from 70% to 35%, mobile phase B increased from 30% to 65%; 15–20 min, mobile phase A 35%, mobile phase B 65%; 20–23 min, mobile phase A increased from 35% to 70%, mobile phase B decreased from 65% to 30%; 23–26 min, mobile phase A 70%, mobile phase B 30%.

[0015] Preferably, the pH of the 0.1% phosphoric acid aqueous solution is 2.2.

[0016] Preferably, the excitation wavelength is 271 nm and the emission wavelength is 503 nm.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides a method for detecting the bacterial quorum sensing signal molecule AI-2. The detection method of this invention is simple and highly operable. The detection limit of AI-2 is 0.006 μg / mL, the quantification limit is 0.020 μg / mL, the relative standard deviations of intra-day precision and inter-day repeatability of retention time and peak area are all less than 5.0%, and the average recovery rate is between 65% and 101%. It can realize the accurate quantitative determination of the bacterial quorum sensing signal molecule AI-2. Attached Figure Description

[0019] Figure 1 The high-performance liquid chromatogram of AI-2 in porcine intestinal contents is shown in Example 1.

[0020] Figure 2 The high-performance liquid chromatogram of AI-2 in bovine rumen contents is shown in Example 2.

[0021] Figure 3 The high-performance liquid chromatogram of the standard sample derivatization method in Example 1 is shown.

[0022] Figure 4 High-performance liquid chromatography (HPLC) chromatograms of AI-2 in the same intestinal contents were obtained by different detection methods for Comparative Example 1 and Example 1. Detailed Implementation

[0023] This invention provides a method for detecting the bacterial quorum sensing signal molecule AI-2, comprising the following steps:

[0024] The sample to be extracted is mixed with hydrochloric acid aqueous solution for extraction. After obtaining the extract, the supernatant is collected by centrifugation. The sample to be extracted includes intestinal contents and / or rumen contents.

[0025] After reacting the supernatant with aminosulfonic acid solution, 2,3-diaminonaphthalene solution was added for derivatization. The derivatized product was then centrifuged, and the supernatant was collected for high-performance liquid chromatography (HPLC) detection.

[0026] In this invention, the precursor molecule 4,5-dihydroxy-2,3-pentanedione of the bacterial quorum sensing signal molecule AI-2 is used for derivatization detection, thereby achieving the purpose of detecting the bacterial quorum sensing signal molecule AI-2. Furthermore, this invention also provides a method for detecting the bacterial quorum sensing signal molecule AI-2 using the above-mentioned method for detecting 4,5-dihydroxy-2,3-pentanedione; and even further, this invention provides the application of the above-mentioned method for detecting the bacterial quorum sensing signal molecule AI-2 in the preparation of reagents for detecting 4,5-dihydroxy-2,3-pentanedione.

[0027] In this invention, the sample to be extracted is mixed with an aqueous hydrochloric acid solution for extraction. Before mixing the sample to be extracted with the aqueous hydrochloric acid solution, the collected sample (such as intestinal contents and / or rumen contents) is freeze-dried, ground, and pulverized to obtain the sample to be extracted. The mass-to-volume ratio of the sample to be extracted to the aqueous hydrochloric acid solution is preferably 1:15 to 1:50 g / mL, more preferably 1:17 to 1:45 g / mL, and even more preferably 1:20 to 1:30 g / mL; the concentration of the aqueous hydrochloric acid solution is preferably 0.05 to 0.15 mol / L, more preferably 0.07 to 0.12 mol / L. In this invention, the extraction is preferably performed using a tissue homogenizer, and the number of extractions is preferably 1 to 2 times. The tissue homogenizer extraction program is 30 times / second, repeated 5 times, and each extraction lasts 30 seconds. Because the content of AI-2 in the intestine is low, the present invention utilizes the above-mentioned hydrochloric acid aqueous solution to extract the sample, which not only maximizes the extraction of the bacterial quorum sensing signal molecule AI-2 from the sample, but also ensures the required amount for detection, thereby improving the accuracy of detecting the bacterial quorum sensing signal molecule AI-2. The pretreatment method using hydrochloric acid aqueous solution in this invention is simple and easy to operate.

[0028] In this invention, after the sample to be extracted is mixed with hydrochloric acid aqueous solution, the extract is centrifuged to obtain the supernatant. The centrifugation is preferably carried out at 2-6°C and 11,000-13,000 rpm for 5-15 minutes, and more preferably at 4°C and 12,000 rpm for 10 minutes.

[0029] In this invention, the intestinal contents and / or rumen contents are the intestinal contents and / or rumen contents of livestock, preferably one or more of pigs, cattle, and sheep. The livestock specifically referred to as the rumen contents are preferably cattle and / or sheep.

[0030] In this invention, after reacting the supernatant with an aminosulfonic acid solution, a 2,3-diaminonaphthalene solution is added for derivatization. The preferred volume ratio of the supernatant to the aminosulfonic acid solution to the 2,3-diaminonaphthalene solution is 2:1:2. The preferred concentration of the aminosulfonic acid solution is 70–90 g / L, more preferably 75–85 g / L, and even more preferably 80 g / L. The preferred concentration of the 2,3-diaminonaphthalene solution is 150–300 μg / mL, more preferably 150–250 μg / mL, and even more preferably 180–220 μg / mL, such as 180, 190, 200, 210, or 220 μg / mL. The solvent for the aminosulfonic acid solution is water, and the solvent for the 2,3-diaminonaphthalene solution is a 0.10 mol / L hydrochloric acid solution. This invention does not have specific limitations on the sources of aminosulfonic acid and 2,3-diaminonaphthalene; commercially available products in the art can be used. The reaction is preferably carried out at room temperature for 5-15 minutes, more preferably 7-12 minutes, and even more preferably 10 minutes. The derivatization temperature is preferably 85-95°C, more preferably 87-93°C, and even more preferably 90°C; the derivatization time is preferably 35-45 minutes, more preferably 38-43 minutes, and even more preferably 40 minutes.

[0031] In this invention, after obtaining the derivative, the supernatant is collected by centrifugation and then analyzed by high-performance liquid chromatography (HPLC). The centrifugation is preferably performed at 2–6°C and 11,000–13,000 rpm for 5–15 min, more preferably at 4°C and 12,000 rpm for 10 min. The chromatographic conditions for HPLC detection are as follows: the chromatographic column is a Zorbax SB-C. 18 The column temperature was 30℃, and the injection volume was 20 μL. The mobile phase consisted of mobile phase A (0.1% phosphoric acid aqueous solution) and mobile phase B (acetonitrile), with a flow rate of 0.8 mL / min. The gradient elution program was as follows: 0–4 min, mobile phase A 70%, mobile phase B 30%; 4–15 min, mobile phase A decreased from 70% to 35%, mobile phase B increased from 30% to 65%; 15–20 min, mobile phase A 35%, mobile phase B 65%; 20–23 min, mobile phase A increased from 35% to 70%, mobile phase B decreased from 65% to 30%; 23–26 min, mobile phase A 70%, mobile phase B 30%. The pH of the 0.1% phosphoric acid aqueous solution was preferably 2.2, and the 0.1% phosphoric acid aqueous solution was prepared by mixing phosphoric acid and water at a volume ratio of 1:1000. During high-performance liquid chromatography (HPLC) detection, the excitation wavelength is 271 nm and the emission wavelength is 503 nm. This invention utilizes the above-mentioned HPLC method to detect target analytes with good peak shape and high resolution, thus improving detection accuracy.

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] In the following embodiments, the standard is DPD / AI-2, purchased from Shanghai Beivanta Biotechnology Co., Ltd., product number BK-12125.

[0034] Example 1

[0035] A method for detecting AI-2, a bacterial quorum sensing signal molecule, in porcine intestinal contents, comprises the following steps:

[0036] (1) Sample acquisition: The collected fresh intestinal contents of pigs were frozen at a temperature below -20℃. After the contents were frozen solid, they were freeze-dried using a freeze dryer. After they were completely dry, they were ground and pulverized to obtain a homogeneous sample to be extracted.

[0037] (2) Sample extraction: Weigh 0.05g of the sample to be extracted in step (1), add 12 ceramic grinding beads, and then add 1.0mL of 0.10mol / L hydrochloric acid aqueous solution for tissue grinding and extraction (the grinding and extraction is performed using a tissue grinder, the extraction program is 30 times / second, repeated extraction 5 times, and each extraction is 30s). After extraction once, centrifuge at 12000rpm for 10min at 4℃, and take the supernatant to obtain the sample to be derived.

[0038] (3) Sample derivatization: Mix 400 μL of the sample to be derivatized obtained in step (2) with 200 μL of 80 g / L aminosulfonic acid solution, react at room temperature for 10 min, then add 400 μL of 200 μg / mL 2,3-diaminonaphthalene solution, derivatize at 90℃ for 40 min, centrifuge the derivatized product at 12000 rpm for 10 min at 4℃, take the supernatant, filter the supernatant through a 0.22 μm filter membrane and perform high performance liquid chromatography analysis, where the liquid chromatography conditions are as follows:

[0039] Column: Zorbax SB-C 18 (4.6 × 250 mm, 5 μm, Agilent); Injection volume: 20 μL; Flow rate: 0.8 mL / min; Column temperature: 30 °C; Mobile phase A: 0.1% phosphoric acid aqueous solution, Mobile phase B: acetonitrile. Gradient elution, elution program shown in Table 1, flow rate 0.8 mL / min. Run time: 26 min; Fluorescence detector, excitation wavelength: 271 nm, emission wavelength: 503 nm.

[0040] The 0.1% phosphoric acid aqueous solution is prepared by mixing phosphoric acid and water at a volume ratio of 1:1000 until homogeneous, and the pH of the 0.1% phosphoric acid aqueous solution is 2.2.

[0041] Table 1 Gradient elution program

[0042]

[0043]

[0044] The chromatogram of AI-2 in porcine intestinal contents detected by high performance liquid chromatography is shown below. Figure 1 The retention time is 9.235 min.

[0045] Example 2

[0046] A method for detecting the bacterial quorum sensing signal AI-2 in bovine rumen contents, comprising the following steps:

[0047] (1) Sample acquisition: Fresh bovine rumen contents were collected and frozen at a temperature below -20℃. After the contents were frozen solid, they were freeze-dried using a freeze dryer. After they were completely dry, they were ground and pulverized to obtain a homogeneous sample to be extracted.

[0048] (2) Sample extraction: Weigh 0.05g of the sample to be extracted in step (1), add 12 ceramic grinding beads, and then add 1.0mL of 0.10mol / L hydrochloric acid aqueous solution for the first tissue grinding extraction (the tissue grinding extraction is performed using a tissue grinder, the extraction program is 30 times / second, repeated extraction 5 times, and each extraction is 30 seconds), centrifuge at 12000rpm for 10min at 4℃, and take the supernatant a and the filter residue a; add 1.0mL of 0.10mol / L hydrochloric acid aqueous solution to the filter residue a, and then perform the second tissue grinding extraction (the tissue grinding extraction is performed using a tissue grinder, the extraction program is 30 times / second, repeated extraction 5 times, and each extraction is 30 seconds), centrifuge at 12000rpm for 10min at 4℃, and take the supernatant b. Combine the supernatant a and the supernatant b to obtain the sample to be derived.

[0049] (3) Sample derivatization determination: 400 μL of the sample to be derivatized obtained in step (2) was mixed with 200 μL of 80 g / L aminosulfonic acid solution and reacted at room temperature for 10 min. Then, 400 μL of 200 μg / mL 2,3-diaminonaphthalene solution was added, and the derivatization was carried out at 90℃ for 40 min. The derivatized product was centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was collected and filtered through a 0.22 μm filter membrane for high performance liquid chromatography analysis. The liquid chromatography conditions were as follows:

[0050] Column: Zorbax SB-C 18(4.6 × 250 mm, 5 μm, Agilent); Injection volume: 20 μL; Flow rate: 0.8 mL / min; Column temperature: 30 °C; Mobile phase A: 0.1% phosphoric acid aqueous solution, Mobile phase B: acetonitrile. Gradient elution, elution program shown in Table 1, flow rate 0.8 mL / min. Run time: 26 min; Fluorescence detector, excitation wavelength: 271 nm, emission wavelength: 503 nm.

[0051] The 0.1% phosphoric acid aqueous solution is prepared by mixing phosphoric acid and water at a volume ratio of 1:1000 until homogeneous, and the pH of the 0.1% phosphoric acid aqueous solution is 2.2.

[0052] The chromatogram of AI-2 in bovine rumen contents detected by high performance liquid chromatography is shown below. Figure 2 The retention time was 9.217 min.

[0053] Example 3

[0054] Determination of detection limit and quantitation limit

[0055] A standard stock solution of DPD / AI-2 with a concentration of 1.00 mg / mL was used as the stock solution. A standard curve was prepared using 0.10 mol / L HCl as the diluent. The sample derivatization method described in Example 1 was followed for detection. The target compound was further diluted based on the peak response intensity before derivatization to determine its limit of quantitation (LOQ) and limit of detection (LOD). A linear regression equation was obtained by plotting the target compound concentration on the x-axis and the peak area after derivatization on the y-axis. The LOD was set at a concentration with a signal-to-noise ratio of 3 times and the LOQ at a concentration with a signal-to-noise ratio of 10 times. The results are shown in Table 2.

[0056] Table 2 Results of Limit of Quantitation and Limit of Detection

[0057]

[0058] The chromatograms of the standards detected by high-performance liquid chromatography are shown below. Figure 3 In the spectrum, DAN represents 2,3-diaminonaphthalene solution, and DAN+DPD represents DAN-modified standard with a retention time of 9.230 min.

[0059] According to the results in Table 2, the detection limit of AI-2 in intestinal contents detected by the detection method in Example 1 was 0.006 μg / mL, and the quantitation limit was 0.020 μg / mL.

[0060] Example 4

[0061] Precision and repeatability determination

[0062] Following the sample derivatization method of Example 1, the standard sample was measured in parallel six times on the same day (n=6). The mean concentration (Mean) and standard deviation (SD) of the measured results were calculated, and the precision (CV%) was calculated as SD ÷ Mean × 100. The coefficient of variation was calculated. Then, the measurements were performed for three consecutive days (n=18), and the mean concentration (Mean) and standard deviation (SD) of the measured results were calculated. The repeatability (CV%) was calculated as SD ÷ Mean × 100. The results are shown in Tables 3 and 4 below.

[0063] Table 3 Precision of Standard Samples

[0064]

[0065]

[0066] Table 4. Repeatability of Standards

[0067]

[0068] The results in Tables 3 and 4 show that the precision and repeatability of the sample derivatization method of the present invention for determining AI-2 are good.

[0069] Example 5

[0070] Determination of the average recovery rate of the detection methods in Examples 1 and 2

[0071] Using intestinal contents samples as test samples, 10 μL, 20 μL, and 40 μL of standard solution at 10 μg / mL were added to each intestinal contents sample, respectively, with final concentrations of 0.100 μg / mL, 0.200 μg / mL, and 0.400 μg / mL. Each treatment was repeated 6 times, and the spiked recovery rate of the samples was determined according to the detection method in Example 1.

[0072] Using rumen contents samples as test samples, 20 μL, 40 μL, and 80 μL of standard solution at 10 μg / mL were added to each rumen contents sample, respectively, with final concentrations of 0.100 μg / mL, 0.200 μg / mL, and 0.400 μg / mL. Each treatment was repeated 6 times, and the spiked recovery rate of the samples was determined according to the detection method in Example 2.

[0073] Spiked recovery rate (%) = (measured value - initial value) ÷ spiked value × 100.

[0074] Table 5. Spike recovery results for intestinal contents and rumen contents samples.

[0075]

[0076] The results in Table 5 show that the average recovery rate of AI-2 in intestinal contents samples detected by the detection method of the present invention is 82% to 101%, and the average recovery rate of AI-2 in rumen contents samples detected by the detection method of the present invention is 65% to 74%.

[0077] Comparative Example 1

[0078] The difference between this comparative example and Example 1 is that the mobile phase A in this comparative example is 0.1% formic acid aqueous solution, and the mobile phase B is acetonitrile. The 0.1% formic acid aqueous solution is prepared by mixing formic acid and water at a volume ratio of 1:1000, and the pH is 2.65. The other detection methods in this comparative example are the same as those in Example 1.

[0079] Figure 4 The results showed that, compared with the comparative chromatogram (the resolution of the detected DPD chromatographic peak was 0.71, and the chromatographic peak was a single peak), the resolution of the DPD chromatographic peak obtained by the detection method of Example 1 was 2.46. The chromatographic peak resolution of the detection method of the present invention was improved, and the measurement results were more accurate.

[0080] Example 6

[0081] A method for detecting the bacterial quorum sensing signal AI-2 in porcine intestinal contents, comprising the following steps:

[0082] (1) Sample acquisition: The collected fresh intestinal contents of pigs were frozen at a temperature below -20℃. After the contents were frozen solid, they were freeze-dried using a freeze dryer. After they were completely dry, they were ground and pulverized to obtain a homogeneous sample to be extracted.

[0083] (2) Sample extraction: Weigh 0.02g of the sample to be extracted in step (1), add 12 ceramic grinding beads, and then add 1.0mL of 0.10mol / L hydrochloric acid aqueous solution for tissue grinding and extraction (the grinding and extraction is performed using a tissue grinder, the extraction program is 30 times / second, repeated extraction 5 times, and each extraction is 30s). After extraction once, centrifuge at 12000rpm for 10min at 4℃, and take the supernatant to obtain the sample to be derived.

[0084] (3) Sample derivatization: Mix 400 μL of the sample to be derivatized obtained in step (2) with 200 μL of 80 g / L aminosulfonic acid solution, react at room temperature for 10 min, then add 400 μL of 200 μg / mL 2,3-diaminonaphthalene solution, derivatize at 90℃ for 40 min, centrifuge the derivatized product at 12000 rpm for 10 min at 4℃, take the supernatant, filter the supernatant through a 0.22 μm filter membrane and perform high performance liquid chromatography analysis, where the liquid chromatography conditions are as follows:

[0085] Column: Zorbax SB-C 18 (4.6 × 250 mm, 5 μm, Agilent); Injection volume: 20 μL; Flow rate: 0.8 mL / min; Column temperature: 30 °C; Mobile phase A: 0.1% phosphoric acid aqueous solution, Mobile phase B: acetonitrile. Gradient elution, elution program shown in Table 1, flow rate 0.8 mL / min. Run time: 26 min; Fluorescence detector, excitation wavelength: 271 nm, emission wavelength: 503 nm.

[0086] The 0.1% phosphoric acid aqueous solution is prepared by mixing phosphoric acid and water at a volume ratio of 1:1000 until homogeneous, and the pH of the 0.1% phosphoric acid aqueous solution is 2.2.

[0087] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting bacterial quorum-sensing signal molecule AI-2, characterized by, Includes the following steps: The sample to be extracted is mixed with hydrochloric acid aqueous solution for extraction. After obtaining the extract, the supernatant is collected by centrifugation. The sample to be extracted includes pig intestinal contents and / or bovine rumen contents. After reacting the supernatant with aminosulfonic acid solution, 2,3-diaminonaphthalene solution was added for derivatization. After obtaining the derivatized product, the supernatant was collected by centrifugation and detected by high performance liquid chromatography. The chromatographic conditions in the high performance liquid chromatography detection are as follows: the chromatographic column is Zorbax SB-C 18 , the column temperature is 30 DEG C, the injection volume is 20 mu L; the mobile phase is mobile phase A and mobile phase B, the mobile phase A is 0.1% phosphoric acid aqueous solution, the mobile phase B is acetonitrile, the flow rate is 0.8 mL / min; the gradient elution program is as follows: 0-4 min, the mobile phase A is 70%, the mobile phase B is 30%; 4-15 min, the mobile phase A decreases from 70% to 35%, the mobile phase B increases from 30% to 65%. 15–20 min, mobile phase A is 35%, mobile phase B is 65%; 20–23 min, mobile phase A increases from 35% to 70%, mobile phase B decreases from 65% to 30%; 23–26 min, mobile phase A is 70%, mobile phase B is 30%; fluorescence detector, excitation wavelength: 271 nm, emission wavelength: 503 nm; The mass-to-volume ratio of the sample to be extracted to the hydrochloric acid aqueous solution is 1:15 to 1:50 g / mL; the concentration of the hydrochloric acid aqueous solution is 0.05 to 0.15 mol / L. Before the sample to be extracted is mixed with hydrochloric acid aqueous solution, the process also includes freeze-drying, grinding and pulverizing the collected sample to obtain the sample to be extracted. The collected samples were pig intestinal contents and / or bovine rumen contents.

2. The detection method according to claim 1, characterized in that, The extraction is performed 1 to 2 times.

3. The detection method according to claim 1, characterized in that, The volume ratio of the supernatant, aminosulfonic acid solution, and 2,3-diaminonaphthalene solution is 2:1:2; the concentration of the aminosulfonic acid solution is 70–90 g / L; and the concentration of the 2,3-diaminonaphthalene solution is 150–300 μg / mL.

4. The detection method according to claim 1, characterized in that, The reaction is carried out at room temperature for 5 to 15 minutes.

5. The detection method according to claim 1, characterized in that, The derivatization temperature is 85–95°C, and the derivatization time is 35–45 min.

6. The detection method according to claim 1, characterized in that, The pH of the 0.1% phosphoric acid aqueous solution is 2.2.

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