Primer pair for identifying bezoar-based antigen and application of primer pair

Abstract

The invention relates to a primer pair for identifying bezoar primordium. The primer pair is selected from any one group of primer pairs as follows: a primer pair 1: a forward primer has a nucleotide sequence as shown in SEQ ID NO: 1, and a reverse primer has a nucleotide sequence as shown in SEQ ID NO: 2; and in the primer pair 2, the nucleotide sequence of the forward primer is shown as SEQ ID NO: 3, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO: 4. The primer pair can be used for effectively identifying the calculus bovis-based antigen, and has relatively good specificity, sensitivity and anti-interference performance.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine identification technology, specifically relating to a primer pair for identifying the origin of bezoar and its application. Background Technology

[0002] Natural bezoar is a pathological product of cattle. Due to its extremely limited natural production, it cannot meet market demand. Therefore, artificial substitutes for natural bezoar have been prepared, mainly falling into two categories: one is in vitro cultured bezoar made from fresh bovine bile as the mother liquor, with the addition of deoxycholic acid, cholic acid, and compound bilirubin calcium; the other is artificial bezoar processed from bovine bile powder, cholic acid, porcine deoxycholic acid, bilirubin, and other raw materials. Currently, the sources of bezoar products on the market are quite chaotic, with many cases of substances such as porcine bile being passed off as natural bezoar.

[0003] Origin identification is a method that uses morphological and taxonomic knowledge of plants, animals, or minerals to identify the source of "traditional Chinese medicine" and determine its correct botanical, zoological, or mineral names to ensure the accuracy of the varieties used. Currently, methods for identifying traditional Chinese medicine include traditional methods such as source identification, morphological identification, microscopic identification, and physicochemical identification, as well as modern DNA molecular identification methods.

[0004] However, because bezoar is a dried, calcified stone-like sample with severely damaged cell structure and highly degraded and extremely low DNA content, it cannot provide a sufficient and complete template for molecular identification. Furthermore, the sample is rich in impurities such as bilirubin, bile acids, and calcium salts, which strongly interfere with enzymatic reactions such as PCR. Therefore, to date, no effective DNA molecular identification technique has been established for the identification of bezoar's origin. For example, patent CN106480206A discloses a primer pair for identifying beef, but due to the presence of more impurities and other interfering substances in bezoar DNA samples, this primer pair cannot be used for bezoar identification. CN107881243A discloses a primer pair for identifying bovine components, but this primer pair also suffers from insufficient anti-interference ability and poor sensitivity in the PCR identification of bezoar. Summary of the Invention

[0005] To address the aforementioned technical problems, this application provides a primer pair for identifying bezoar progeny. The primer pair can effectively identify bezoar progeny and has good specificity, sensitivity, and anti-interference properties.

[0006] According to one aspect of this application, a primer pair for identifying bezoar progeny is provided, said primer pair being selected from any of the following sets of primer pairs: Primer pair 1: The nucleotide sequence of the forward primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 2; Primer pair 2: The nucleotide sequence of the forward primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 4.

[0007] According to another aspect of this application, a kit for identifying bovine gallstones is provided, the kit containing the primer pairs described above.

[0008] Optionally, the kit may also contain DNA extraction reagents and PCR amplification reagents.

[0009] According to another aspect of this application, the application of the above primer pairs and the above kit in the identification of bovine gallstones is provided.

[0010] According to another aspect of this application, a method for identifying bovine gallstones is provided, the method comprising the following steps: (1) The DNA sample of the test sample is amplified using the primers described in claim 1; (2) Based on the amplification results, determine whether the sample to be tested is bezoar.

[0011] Optionally, step (2) is: Electrophoretic analysis of the amplification products revealed that the sample showing a specific band was bezoar.

[0012] Optionally, step (2) is: The amplified product was sequenced, and the sample with the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6 was bezoar.

[0013] According to another aspect of this application, a method for extracting bovine blood DNA is provided, the method comprising the step of synergistically lysing the sample using a buffer 1 containing CDAB and a buffer 2 containing SDS.

[0014] Optionally, the synergistic lysis of the sample specifically includes the following steps: (1) Take a sample of bezoar and grind it; (2) Add buffer 1 and incubate; (3) Add buffer 2 and incubate.

[0015] Optionally, the ratio of bezoar sample to buffer 1 to buffer 2 is 30-150 mg : 600-1000 µL : 100-300 µL.

[0016] Optionally, the content of CTBA in buffer 1 is 0.5-3% and the content of SDS in buffer 2 is 8-12%.

[0017] Optionally, the extraction method further includes a DNA magnetic bead separation step after sample lysis, wherein the magnetic beads have a particle size of 300-600 nm.

[0018] According to another aspect of this application, a method for extracting and identifying the origin of bezoar DNA is provided, the method comprising the following steps: (A) Extracting DNA samples from the test sample: DNA samples were extracted using any of the extraction methods described above. (B) Identification of the original bovine gallstone: The method described in any of the above methods shall be used for identification of bovine gallstone origin.

[0019] The positive and beneficial effects achieved by this application are as follows: The primer pairs and their method for identifying the origin of bezoar provided in this application are effective for identifying the origin of bezoar and have good specificity and sensitivity. Using the primer pairs of this application for bezoar origin identification is accurate, reliable, highly sensitive, simple, and saves time and effort.

[0020] The DNA extraction method provided in this application effectively solves the problems of low DNA content and high impurity content in bezoar. Attached Figure Description

[0021] Figure 1 The specificity identification effect of each primer pair on samples from cattle and pigs is shown in the figure. Figure 2 The results show the anti-interference test results of each primer pair for the identification of bezoar; Figure 3 Electrophoresis diagram of the sensitivity effect for identifying N206F / R bezoar; Figure 4 Electrophoresis diagram of the sensitivity effect for identifying N154F / R bezoar; Figure 5 Electrophoresis diagram of the sensitivity effect for identifying N100F / R bezoar; Figure 6 A qPCR graph illustrating the sensitivity of N154F / R bezoar identification; Figure 7 A qPCR graph showing the sensitivity of N100F / R bezoar identification. Detailed Implementation

[0022] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0023] In this application, the use of conventional methods to extract DNA refers to the use of the magnetic bead method animal tissue genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. for DNA extraction, and the specific method is performed in accordance with the product instructions.

[0024] In this application, using a specific method to extract DNA means using the following methods to extract DNA: 1. Reagent preparation ①LA: 100 mM Tris-HCl (pH 8.0), 100 mM EDTA (pH 8.0), 2% Tween-20, 1.4M NaCl, 2% CTAB, add 0.3% β-mercaptoethanol before use.

[0025] ②LB: 10% SDS.

[0026] ③CB: 3M NaAC (sodium acetate).

[0027] ④PA: 25 mM EDTA (PH 8.0), 100 mM Tris-HCl (PH 8.0), 200 mM NaCl, 2% PVP-40.

[0028] ⑤PW1: 10-50 mM Tris-HCl (pH 7.5), 1M NaCl, 25% anhydrous ethanol.

[0029] ⑥PW2: 75% anhydrous ethanol.

[0030] 2. DNA extraction steps ① Take 60mg of sample, rinse twice with PA, add the sample to a 2mL centrifuge tube, add 2 steel balls and grind in a ball mill for 30s; ② Add 800 μL of lysis buffer LA (with 0.3% β-thioethanol), then add 20 μL of proteinase K (vacuum), vortex mix for 30 seconds, and incubate at 37°C for 30 minutes. ③ Add 200 μL of lysis buffer (LB aspiration) and incubate at 65°C for 2 hours; ④ Centrifuge at 12000 rpm for 5 minutes, and transfer the supernatant to a new centrifuge tube; ⑤ Add 800 μL of ice-cold isopropanol and 80 μL of CB, as well as 10 μL of 500 nm magnetic beads (vortex mix), and place at 20 °C for 30 min. ⑥ Magnetic separation to remove the supernatant; ⑦ Add 600 μL PW1 for washing, vortex mix for 30 s, magnetically separate, and remove the supernatant; ⑧ Add 600 μL PW2 for washing, vortex mix for 30 seconds, magnetically separate, remove the supernatant, and air dry in the clean bench with the lid open. 9. Add 50 μL of double-distilled water (vacuum), let stand at room temperature for 5 min, perform magnetic separation, and transfer the supernatant to a new centrifuge tube.

[0031] In this application, the PCR detection kit used is the 2X M5HiPer plus Taq HiFi PCR mix kit from Beijing Polymer Biotechnology Co., Ltd., and the detection method is performed in accordance with the product instructions.

[0032] In this application, the qPCR detection uses the SYBR Green I dye method, and the steps are as follows: 1. Prepare the PCR reaction solution on ice in a 96-well plate or PCR tube according to the following components.

[0033] 2. Perform qPCR according to the following procedure. Stage 1 prevariation: Reps: 1, 95℃ for 30 seconds Stage 2: PCR reaction Reps: 40, 95℃ for 5 seconds, 60℃ for 34 seconds Stage 3: Reps: 1, 95℃ for 15 seconds, 60℃ for 1 minute, 95℃ for 15 seconds The primer sequences and their PCR amplification procedures involved in this application are shown in Table 1 below: Table 1 Primer sequences and their PCR amplification procedures

[0034] Example 1: DNA Sample Preparation DNA was extracted from beef tissue using conventional methods, and the resulting sample was named DNA sample A.

[0035] DNA was extracted from pork tissue using conventional methods, and the resulting sample was named DNA sample B.

[0036] DNA was extracted from bezoar using conventional methods and the resulting sample was named DNA sample C; DNA was extracted using specific methods and the resulting sample was named DNA sample D.

[0037] The concentrations of DNA samples C and D were detected using Qubit, and the A260 / A280 values ​​of DNA samples C and D were detected using Nanodrop. The results are shown in Table 2 below, indicating that the concentration and purity of DNA samples extracted using the specific method are significantly better than those extracted using conventional methods.

[0038] Table 2. Concentration and purity test results of bovine blood DNA extracted by different methods.

[0039] Example 2: Specificity identification effect of each primer pair on samples from cattle DNA sample A and DNA sample B were used as amplification templates. Primer pairs N206F / R, N154F / R, N131F / R, N133F / R, N500F / R, and N100F / R were used for amplification to determine whether each primer pair could specifically identify samples from cattle. The amplification procedure was performed as shown in Table 1. The amplification products of each primer pair were analyzed by agarose gel electrophoresis, and the results are as follows: Figure 1 As shown, lanes 1-12 represent the electrophoresis results of the amplification products of N206F / R (DNA sample A as template), N206F / R (DNA sample B as template), N154F / R (DNA sample A as template), N154F / R (DNA sample B as template), N131F / R (DNA sample A as template), N131F / R (DNA sample B as template), N133F / RF / R (DNA sample A as template), N133F / RF / R (DNA sample B as template), N500F / R (DNA sample A as template), N500F / R (DNA sample B as template), N100F / R (DNA sample A as template), and N100F / R (DNA sample B as template), respectively.

[0040] The results show (see) Figure 1When DNA sample A was used as a template, N206F / R, N154F / R, N500F / R, and N100F / R produced specific bands in the amplification results. However, when DNA sample B was used as a template, no bands were observed. This indicates that N206F / R, N154F / R, N500F / R, and N100F / R can specifically amplify DNA samples from cattle. N131F / R, regardless of whether DNA sample A or DNA sample B was used as a template, produced multiple disordered bands. While N133F / R produced a corresponding band when DNA sample A was used as a template, it was not unique, and when DNA sample B was used as a template, it also produced multiple disordered bands. Therefore, N131F / R and N133F / R cannot specifically identify samples from cattle.

[0041] Example 3: Determination of the anti-interference ability of each primer pair for the identification of low-quality bezoar DNA samples DNA sample C was used as the amplification template, and primer pairs N206F / R, N154F / R, N131F / R, N133F / R, N500F / R, and N100F / R were used for amplification to detect whether each primer pair could specifically identify the bezoar DNA sample. The amplification procedure was performed according to Table 1. The amplification products of each primer pair were analyzed by agarose gel electrophoresis, and the results are as follows. Figure 2 As shown, lanes 1-6 are the electrophoresis results of the amplification products of N206F / R, N154F / R, N131F / R, N133F / R, N500F / R, and N100F / R, respectively.

[0042] The results show (see) Figure 2 Using DNA sample C with low concentration and poor purity as a template, amplification with primers N206F / R and N154F / R yielded obvious specific bands, especially N206F / R, whose band was the brightest and clearest, indicating that N206F / R and N154F / R have good anti-interference properties and can specifically identify bezoar samples. The amplification products of N206F / R and N154F / R were sequenced, and the sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0043] Electrophoresis results for N131F / R and N133F / R showed diffuse bands, indicating that they could not specifically identify bezoar. N500F / R showed almost no bands, indicating that it had very poor anti-interference ability and could not effectively identify bezoar DNA samples. Although N100F / R showed specific bands, the band brightness was very weak, indicating that N100F / R had poor anti-interference ability.

[0044] Example 4: Sensitivity determination of each primer pair for bezoar identification DNA sample D was diluted 10 times sequentially.1 10 2 10 3 10 4 10 5 10 6 Using the diluted samples as templates, amplification was performed using primers N206F / R, N154F / R, N500F / R, and N100F / R, respectively. The results showed that N206F / R could identify up to 10... 4 Diluent (see) Figure 3 N154F / R can identify up to 10 3 Diluent (see) Figure 4 The N500F / R could not identify any diluents, and the N100F / R could only identify up to 10. 1 Diluent, 10 2 The identification bands are extremely weak or almost nonexistent (see [reference]). Figure 5 The above results indicate that N206F / R has the best sensitivity, followed by N154F / R.

[0045] The sensitivity of N206F / R, N154F / R, and N100F / R to bezoar was further detected by qPCR. The results are shown in Table 3 below, where the qPCR chromatogram of N154F / R is shown in the figure. Figure 6 The qPCR diagram of N100F / R is shown below. Figure 7 As shown in the figure. The qPCR results also indicate that N206F / R has the best sensitivity, followed by N154F / R.

[0046] Table 3. qPCR detection results for each primer pair

[0047] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A primer pair for identifying the precursor of bezoar, characterized in that, The primer pairs are selected from any of the following sets of primer pairs: Primer pair 1: The nucleotide sequence of the forward primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 2; Primer pair 2: The nucleotide sequence of the forward primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:

4.

2. A reagent kit for identifying bezoar precursor, characterized in that, The kit contains the primer pair as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also contains DNA extraction reagents and PCR amplification reagents.

4. The use of the primer pair according to claim 1 and the kit according to any one of claims 2-3 in the identification of bovine progeny.

5. A method for identifying the original form of bezoar, characterized in that, The method includes the following steps: (1) The DNA sample of the test sample is amplified using the primers described in claim 1; (2) Based on the amplification results, determine whether the sample to be tested is bezoar.

6. The method according to claim 5, characterized in that, Step (2) is as follows: Electrophoretic analysis of the amplification products revealed that the sample showing a specific band was bezoar.

7. The method according to claim 5, characterized in that, Step (2) is as follows: The amplified product was sequenced, and the sample with the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6 was bezoar.

8. A method for extracting bezoar DNA, characterized in that, The extraction method includes the step of synergistically lysing the sample using buffer 1 containing CDAB and buffer 2 containing SDS.

9. The extraction method according to claim 8, characterized in that, The synergistic lysis of the sample specifically includes the following steps: (1) Take a sample of bezoar and grind it; (2) Add buffer 1 and incubate; (3) Add buffer 2 and incubate.

10. The extraction method according to claim 8, characterized in that, The ratio of bezoar sample to buffer 1 to buffer 2 is 30-150 mg : 600-1000 µL : 100-300 µL.

11. The extraction method according to claim 8, characterized in that, The CTBA content in buffer 1 is 0.5-3%; the SDS content in buffer 2 is 8-12%.

12. The extraction method according to claim 8, characterized in that, The extraction method further includes a DNA magnetic bead separation step after sample lysis, wherein the magnetic beads have a particle size of 300-600 nm.

13. A method for extracting and identifying the origin of bezoar DNA, characterized in that, The method includes the following steps: (A) Extracting DNA samples from the test sample: DNA sample extraction was performed using the extraction method described in any one of claims 8-12; (B) Identification of the original bovine gallstone: The method described in any one of claims 5-7 shall be used for the identification of bovine gallstone origin.

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