A multiplex specific PCR identification method for extracts of Bombyx batryticatus and fried Bombyx batryticatus and its application in identification of traditional Chinese medicines

Abstract

The present invention discloses a primer for multiple PCR identification of Bombyx mori and / or fried Bombyx mori and their decoction pieces, water extract products and formula granules, comprising a first primer pair and a second primer pair; wherein the sequence of the upstream primer of the first primer pair is shown as SEQ ID NO: 1, and the sequence of the downstream primer thereof is shown as SEQ ID NO: 2; the sequence of the upstream primer of the second primer pair is shown as SEQ ID NO: 3, and the sequence of the downstream primer thereof is shown as SEQ ID NO: 4. The present invention also discloses the application of the above primers, a kit based on the primers and an identification method based on the primers. The implementation of the present invention can effectively identify the authenticity of Bombyx mori and / or fried Bombyx mori and their decoction pieces, water extract products and formula granules.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine identification, and in particular to a multiple specific PCR identification method of silkworm pupa and fried silkworm pupa extracts and application thereof in identification of traditional Chinese medicines. Background Art

[0002] The 2020 edition of the Chinese Pharmacopoeia stipulates that the source of the silkworm is the dried body of the 4-5 instar larvae of the silkworm Bombyx mori Linnaeus, an insect of the Bombycidae family, infected (or artificially inoculated) with Beauveria bassiana (Bals.) Vuillant and killed. It is mostly produced in spring and autumn, and the silkworms that died of infection with Beauveria bassiana are dried to obtain the silkworm. As a commonly used Chinese herbal medicine animal medicine, the silkworm is used in large quantities. There are many counterfeits or adulterated products on the market, such as silkworms killed by green muscardine infection, dead silkworms dyed white with lime water or wrapped in Beauveria bassiana spore powder, and black dead silkworms. They have similar properties and morphology. It is difficult to rely on traditional identification methods and the accuracy is low, especially after the processed products made by bran frying, water extraction to make standard decoctions, and further processed into formula granules, after losing the medicinal properties, it is even more difficult to effectively identify. In order to ensure the quality and efficacy of the medicinal materials of silkworm, a method that can accurately and quickly distinguish the genuine silkworm from its counterfeits is urgently needed.

[0003] At present, Chinese patent CN106048020A discloses a gene ID card of an animal medicinal material, Bombyx mori, which can identify silkworms based on DNA barcode technology of COI and ITS sequences, and constructs a neighbor-joining (NJ) tree based on ITS sequences to distinguish Beauveria from other pathogenic strains. Since this method is based on COI and ITS universal primers, the sequencing results need to be processed and compared after the PCR product is sequenced, and the operation steps are cumbersome and time-consuming, and it is impossible to quickly detect the authenticity of Bombyx mori. In addition, Chinese patent CN107164471A discloses a molecular identification method for quickly identifying the authenticity of Beauveria in the Chinese medicinal material Bombyx mori, which is based on the mitochondrial full genes of Beauveria and multiple Cordyceps species, designs specific primers for Beauveria, and realizes the identification of the authenticity of Beauveria in Bombyx mori by PCR reaction. However, the above method is limited to Bombyx mori medicinal materials and has a small scope of application. At present, the specific primers, kits and identification methods used to identify Bombyx batryticatus are mainly used for medicinal materials, while there are few studies on the specific identification of Bombyx batryticatus and its processed products, fried Bombyx batryticatus, water extracts and formula granules, and it is not possible to simultaneously identify Bombyx mori and Beauveria bassiana in Bombyx batryticatus and fried Bombyx batryticatus through the same amplification system and procedure. Therefore, in order to ensure the safety of use and clinical efficacy of Chinese medicine formula granules, it is necessary to establish an identification method with high accuracy and good specificity. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a primer for multiplex PCR identification of silkworm pupa and / or fried silkworm pupa and their decoction pieces, water extract products and formula granules, which can effectively identify the authenticity of silkworm pupa and / or fried silkworm pupa and their decoction pieces, water extract products and formula granules.

[0005] The technical problem that the present invention needs to solve is to provide the application of primers for multiple PCR identification of the silkworm and / or fried silkworm and their decoction pieces, water extract products and formula granules.

[0006] The technical problem that the present invention also solves is to provide a kit that can quickly identify the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules.

[0007] The technical problem that the present invention also solves is to provide a method for quickly identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules, which has good accuracy and high specificity.

[0008] In order to solve the above technical problems, the present invention provides a primer for multiple PCR identification of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules, which comprises a first primer pair and a second primer pair;

[0009] The sequence of the upstream primer of the first primer pair is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2;

[0010] The sequence of the upstream primer of the second primer pair is shown in SEQ ID NO:3, and the sequence of the downstream primer thereof is shown in SEQ ID NO:4.

[0011] The specific sequences of the first primer pair and the second primer pair are shown in the following table:

[0012]

[0013] In order to solve the above technical problems, the present invention provides the use of primers for multiple PCR identification of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules in (1), (2), (3) or (4):

[0014] (1) Application in identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules;

[0015] (2) Application in identifying the authenticity of Chinese patent medicines containing Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, aqueous extracts and granules;

[0016] (3) preparing a kit for identifying Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, aqueous extract products and formulated granules;

[0017] (4) Application in constructing a method for rapidly identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules.

[0018] In one embodiment, the application of identifying the authenticity of Bombyx mori and / or stir-fried Bombyx mori and their decoction pieces, water extract products and formula granules is to simultaneously identify whether Bombyx mori and / or stir-fried Bombyx mori and their decoction pieces, water extract products and formula granules are derived from Bombyx mori and Beauveria bassiana. If Bombyx mori and / or stir-fried Bombyx mori and their decoction pieces, water extract products and formula granules are derived from Bombyx mori and Beauveria bassiana at the same time, the Bombyx mori and / or stir-fried Bombyx mori and their decoction pieces, water extract products and formula granules are authentic; otherwise, they are counterfeit and inferior products.

[0019] In order to solve the above technical problems, the present invention provides a kit, which comprises a first primer pair and a second primer pair;

[0020] The sequence of the upstream primer of the first primer pair is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2;

[0021] The sequence of the upstream primer of the second primer pair is shown in SEQ ID NO:3, and the sequence of the downstream primer thereof is shown in SEQ ID NO:4.

[0022] In one embodiment, the kit also includes a DNA positive control, a PCR reaction system, and an electrophoresis identification system.

[0023] In order to solve the above technical problems, the present invention provides a method for quickly identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules, comprising the following steps:

[0024] S1, extracting genomic DNA of the sample to be tested;

[0025] S2, using the genomic DNA as a template and the primers as claimed in claim 1 to perform PCR amplification to obtain an amplified product;

[0026] S3. Performing agarose gel electrophoresis analysis on the amplified product to determine the authenticity of the sample to be tested.

[0027] In one embodiment, in step S1, the genomic DNA of the sample to be tested is extracted by the following method:

[0028] Take the sample to be tested, add CTAB precipitation solution for precipitation extraction 1 to 3 times; take the precipitate, add CTAB extraction solution, proteinase K, and β-mercaptoethanol for extraction, and then add chloroform-isoamyl alcohol for extraction 1 to 3 times; take the supernatant after extraction, add isopropanol or isopropanol-sodium acetate for precipitation extraction, wash and incubate the precipitate, and then dissolve it in water to obtain the genomic DNA of the sample to be tested.

[0029] In one embodiment, step S2 includes:

[0030] The genomic DNA is used as a template and the first primer pair and the second primer pair are used to perform PCR amplification to obtain an amplified product.

[0031] In one embodiment, in step S2, the PCR amplification procedure is: pre-denaturing the amplification system at a temperature of 94-96°C for 4-6 minutes, then cycling 38-45 times under a preset program, and finally extending at 71-75°C for 4-6 minutes;

[0032] The preset program is: first denaturation at a temperature of 94-96°C for 20-40s, then annealing at a temperature of 50-56°C for 20-40s, and then extension at a temperature of 65-80°C for 20-40s.

[0033] In one embodiment, the reaction system for PCR amplification is: 12.5 μL of 2×Taq PCR Mix, 0.3-0.7 μL of the upstream primer of the first primer pair, 0.3-0.7 μL of the downstream primer of the first primer pair, 0.3-0.7 μL of the upstream primer of the second primer pair, 0.3-0.7 μL of the downstream primer of the second primer pair, 0.5-2.5 μL of DNA template, and sterile double distilled water to 25 μL.

[0034] Preferably, the reaction system for the PCR amplification is: 12.5 μL of 2×Taq PCR Mix, 0.5 μL of the upstream primer of the first primer pair, 0.5 μL of the downstream primer of the first primer pair, 0.5 μL of the upstream primer of the second primer pair, 0.5 μL of the downstream primer of the second primer pair, 1 μL of DNA template, and sterile double distilled water to make up to 25 μL.

[0035] In one embodiment, step S3 includes:

[0036] The amplified product is subjected to agarose gel electrophoresis analysis. If the amplified product produces a band at 163 bp, the sample to be tested contains Bombyx mori; if the amplified product produces a band at 260 bp, the sample to be tested contains Beauveria bassiana;

[0037] When the sample to be tested contains both Bombyx mori and Beauveria bassiana, the sample to be tested is authentic; otherwise, the sample to be tested is a counterfeit product.

[0038] The implementation of the present invention has the following beneficial effects:

[0039] 1. The primers provided by the present invention can perform multiple site-specific PCR reactions, and can effectively identify whether Bombyx mori and Beauveria bassiana are contained in Bombyx mori and / or fried Bombyx mori and their decoction pieces, water extract products and formula granules, thereby distinguishing the authenticity of Bombyx mori and / or fried Bombyx mori and their decoction pieces, water extract products and formula granules, and Chinese patent medicines containing Bombyx mori and / or fried Bombyx mori and their decoction pieces, water extract products and formula granules. The present invention takes the decoction pieces, water extract products and formula granules of Bombyx mori and fried Bombyx mori as research objects, designs Bombyx mori and Beauveria bassiana specific primers based on COI gene and ITS gene respectively, realizes the multiple specific PCR method and its specific primers for simultaneously identifying Bombyx mori and Beauveria bassiana through the same amplification system and procedure, and adds the applicability research of Chinese patent medicines containing Bombyx mori to ensure the safety of medication and clinical efficacy of Chinese medicine formula granules, and provides a high-accuracy and good specificity identification method.

[0040] 2. The identification method provided by the present invention has good accuracy and high specificity, and can quickly and accurately identify the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules, and can solve the problem that standard decoctions and Chinese medicine formula granules are difficult to identify after losing their shape. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The electrophoresis diagram of the primer screening of Example 1; wherein, M is a DNA molecular weight standard, 1 is a Bombyx batryticatus formula granule, 2 is a Bombyx batryticatus decoction piece, 3 is a Bombyx batryticatus standard decoction (lyophilized powder), and N is a blank (ddH2O);

[0042] Figure 2 It is the sample suitability result of the multiple specific PCR method of Example 2; wherein, M is a DNA molecular weight standard, 1 is a Bombyx batryticatus control medicinal material, 2-6 are Bombyx batryticatus decoction pieces, 7-12 are Bombyx batryticatus standard decoction (lyophilized powder), 13-18 are Bombyx batryticatus formula granules, 19-24 are stir-fried Bombyx batryticatus decoction pieces, 25-30 are Bombyx batryticatus standard decoction (lyophilized powder), 31-36 are Bombyx batryticatus formula granules, 37 are beetle larvae medicinal materials, 38-41 are silkworm pupa medicinal materials, 41-42 are dead silkworm medicinal materials, 43-44 are Yixian pills, and N is blank (ddH2O);

[0043] Figure 3 The sample suitability results of the multiplex specific PCR method of Example 3 are as follows: wherein, M is a DNA molecular weight standard, 1. Bombyx batryticatus control medicinal material, 2-7. Bombyx batryticatus decoction pieces, 8-13. Bombyx batryticatus standard decoction, 14-19. Bombyx batryticatus formula granules, 20-25. Stir-fried Bombyx batryticatus decoction pieces, 26-31. Stir-fried Bombyx batryticatus standard decoction, 32-37. Stir-fried Bombyx batryticatus formula granules, 38. Scarabaeidae larvae medicinal materials, 39-40. Dead silkworm medicinal materials, 41. Bombyx batryticatus decoction pieces, 42-45. Silkworm medicinal materials not infected with Beauveria bassiana, 46-47. Yidian Pills, and N is blank (ddH2O). DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0045] In order to solve the above technical problems, the present invention provides a primer for multiple PCR identification of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules, which comprises a first primer pair and a second primer pair;

[0046] The sequence of the upstream primer of the first primer pair is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2;

[0047] The sequence of the upstream primer of the second primer pair is shown in SEQ ID NO:3, and the sequence of the downstream primer thereof is shown in SEQ ID NO:4.

[0048] The specific sequences of the first primer pair and the second primer pair are shown in Table 1:

[0049] Table 1 is the information table of the first primer pair and the second primer pair

[0050]

[0051] Accordingly, the present invention provides the use of primers for multiplex PCR identification of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules in (1), (2), (3) or (4):

[0052] (1) Application in identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules;

[0053] (2) Application in identifying the authenticity of Chinese patent medicines containing Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, aqueous extracts and granules;

[0054] (3) preparing a kit for identifying Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, aqueous extract products and formulated granules;

[0055] (4) Application in constructing a method for rapidly identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules.

[0056] In one embodiment, the application of identifying the authenticity of Bombyx mori and / or stir-fried Bombyx mori and their decoction pieces, water extract products and formula granules is to simultaneously identify whether Bombyx mori and / or stir-fried Bombyx mori and their decoction pieces, water extract products and formula granules are derived from Bombyx mori and Beauveria bassiana. If Bombyx mori and / or stir-fried Bombyx mori and their decoction pieces, water extract products and formula granules are derived from Bombyx mori and Beauveria bassiana at the same time, the Bombyx mori and / or stir-fried Bombyx mori and their decoction pieces, water extract products and formula granules are authentic; otherwise, they are counterfeit and inferior products.

[0057] Furthermore, the present invention provides a kit, comprising a first primer pair and a second primer pair;

[0058] The sequence of the upstream primer of the first primer pair is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2;

[0059] The sequence of the upstream primer of the second primer pair is shown in SEQ ID NO:3, and the sequence of the downstream primer thereof is shown in SEQ ID NO:4.

[0060] In one embodiment, the kit also includes a DNA positive control, a PCR reaction system, and an electrophoresis identification system.

[0061] Furthermore, the present invention also provides a method for quickly identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules, comprising the following steps:

[0062] S1, extracting genomic DNA of the sample to be tested;

[0063] In one embodiment, in step S1, the genomic DNA of the sample to be tested is extracted by the following method:

[0064] Take the sample to be tested, add CTAB precipitation solution for precipitation extraction 1 to 3 times; take the precipitate, add CTAB extraction solution, proteinase K, and β-mercaptoethanol for extraction, and then add chloroform-isoamyl alcohol for extraction 1 to 3 times; take the supernatant after extraction, add isopropanol or isopropanol-sodium acetate for precipitation extraction, wash and incubate the precipitate, and then dissolve it in water to obtain the genomic DNA of the sample to be tested.

[0065] Preferably, the genomic DNA of the sample to be tested is extracted by the following method:

[0066] Take the sample to be tested, add CTAB precipitation solution for precipitation extraction twice; take the precipitate, add CTAB extraction solution, proteinase K, and β-mercaptoethanol for extraction, and then add chloroform-isoamyl alcohol for extraction 3 times; take the supernatant after extraction, add isopropanol or isopropanol-sodium acetate for precipitation extraction, wash and incubate the precipitate, and then dissolve it with water to obtain the genomic DNA of the sample to be tested.

[0067] Further, the concentration of the extracted genomic DNA is measured. In one embodiment, a genomic DNA sample is taken, and the DNA concentration is measured using a BioSpec-nano micro-volume UV spectrophotometer, while OD260 / OD230 and OD260 / OD280 are recorded, and the concentration is adjusted to 100-400 ng / μL.

[0068] S2, using the genomic DNA as a template and the primers as claimed in claim 1 to perform PCR amplification to obtain an amplified product;

[0069] In one embodiment, the PCR amplification procedure is: pre-denaturing the amplification system at a temperature of 94-96°C for 4-6 minutes, then cycling 38-45 times under a preset program, and finally extending at 71-75°C for 4-6 minutes;

[0070] The preset program is: first denaturation at 95°C for 30s, then annealing at 53°C for 30s, and then extension at 72°C for 30s.

[0071] Preferably, the PCR amplification program is: pre-denaturing the amplification system at 95°C for 5 minutes, then cycling 41 times under the preset program, and finally extending at 72°C for 5 minutes; wherein the preset program is: first denaturing at 95°C for 30 seconds, then annealing at 53°C for 30 seconds, and then extending at 72°C for 30 seconds. Under this program, the target band brightness is higher.

[0072] In one embodiment, step S2 includes:

[0073] The genomic DNA is used as a template and the first primer pair and the second primer pair are used to perform PCR amplification to obtain an amplified product.

[0074] In one embodiment, the reaction system for PCR amplification is: 10-15 μL of 2×Taq PCR Mix, 0.3-0.7 μL of the upstream primer of the first primer pair, 0.3-0.7 μL of the downstream primer of the first primer pair, 0.3-0.7 μL of the upstream primer of the second primer pair, 0.3-0.7 μL of the downstream primer of the second primer pair, 0.5-2.5 μL of DNA template, and sterile double distilled water to 25 μL.

[0075] Preferably, the reaction system for the PCR amplification is: 12.5 μL of 2×Taq PCR Mix, 0.5 μL of the upstream primer of the first primer pair, 0.5 μL of the downstream primer of the first primer pair, 0.5 μL of the upstream primer of the second primer pair, 0.5 μL of the downstream primer of the second primer pair, 1 μL of DNA template, and sterile double distilled water to make up to 25 μL.

[0076] S3. Performing agarose gel electrophoresis analysis on the amplified product to determine the authenticity of the sample to be tested.

[0077] In one embodiment, 5 μL of 6× loading buffer is added to the amplification product reaction system, mixed, 6 to 8 μL of the mixed product is spotted on a 1.5% agarose gel, electrophoresed at 150 to 170 V for 30 min, developed with GelRed, and finally observed and recorded the results on a gel imager.

[0078] In one embodiment, step S3 includes:

[0079] The amplified product is subjected to agarose gel electrophoresis analysis. If the amplified product produces a band at 163 bp, the sample to be tested contains Bombyx mori; if the amplified product produces a band at 260 bp, the sample to be tested contains Beauveria bassiana;

[0080] When the sample to be tested contains both Bombyx mori and Beauveria bassiana, the sample to be tested is authentic; otherwise, the sample to be tested is a counterfeit product.

[0081] The present invention is described below with reference to specific embodiments.

[0082] Example 1

[0083] Design and screening of primers for multiplex PCR identification of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formulated granules

[0084] (1) Primer design

[0085] The ITS sequences of Cytochrome Oxidase I (CO I) of Bombyx mori, Beauveria bassiana and Metarhizium were searched and downloaded from the GeneBank database. Since the sequence information of the pseudo-bombyx mori scarab beetle larvae was lacking in NCBI, the Bombyx mori primers were designed based on the sequence information of Bombyx mori. The sequences of Bombyx mori, Beauveria bassiana and Metarhizium were homologously aligned using BioEdit software, and the specific SNP sites of Bombyx mori and Beauveria bassiana were analyzed after proofreading. The base sequences containing the SNP sites were imported into Primer Premier5 software for primer design. After preliminary screening, the 6 pairs of primers shown in Table 2 were determined and synthesized by Shanghai Biotech Co., Ltd.

[0086] Table 2 Primer information

[0087]

[0088]

[0089] (2) Primer screening

[0090] The PCR amplification system and procedure for Bombyx mori and Beauveria bassiana were established, as shown in Table 3:

[0091] Table 3 PCR reaction system of Bombyx mori and Beauveria bassiana (25 μL)

[0092]

[0093] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 30 s, for a total of 41 cycles; and finally extension at 72°C for 5 min.

[0094] PCR amplification was performed using the genomic DNA obtained in Example 2 and the primers shown in Table 2. After the PCR amplification reaction was completed, 5 μL of 6× loading buffer was added to the reaction system and mixed. 6 to 8 μL of the mixed product was spotted on a 1.5% agarose gel and electrophoresed at 150 V for 30 min. The gel was developed with GelRed and the results were observed on a gel imager. Figure 1 .

[0095] Depend on Figure 1 It can be seen that the blank control of the primer combination JiaC07.F / R and BJJ03.F / R did not show any reaction bands, indicating that the PCR reaction process was pollution-free, the results were reliable, and the target bands were brighter. The target bands of Bombyx mori and Beauveria bassiana in the decoction pieces could be clearly distinguished. The lengths of the target bands obtained by PCR amplification were 163bp and 260bp, respectively, but the Bombyx mori bands were not obtained for the freeze-dried powder and formula granules of the standard decoction. In addition, the primer combination JiaC08.F / R and BJJ03.F / R can also amplify Bombyx mori and Beauveria bassiana, but the Bombyx mori bands were not obtained for the freeze-dried powder and formula granules of the standard decoction. It shows that the above two sets of primers can simultaneously and specifically detect Bombyx mori and Beauveria bassiana in medicinal materials or decoction pieces. Subsequently, by optimizing the PCR conditions, the annealing temperature was determined to be 53°C and the number of cycles was 41 times, which is suitable for the identification of freeze-dried powder and formula granules of the standard decoction.

[0096] Example 2

[0097] A method for quickly identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules and its applicability test

[0098] S1, extracting genomic DNA of the sample to be tested;

[0099] Prepare the samples to be tested according to the sample list shown in Table 4, and extract genomic DNA. Among them, the fried silkworm pupa is prepared by washing the silkworm pupa and frying it in a hot pan. In addition, the standard decoction is a freeze-dried powder. Yixian Pill is a Chinese patent medicine, and during its preparation, the powdered silkworm or fried silkworm pupa is directly added.

[0100] Table 4 Sample list

[0101]

[0102] The following method was used to extract genomic DNA from samples of Bombyx batryticatus medicinal materials, Bombyx batryticatus decoction pieces, stir-fried Bombyx batryticatus decoction pieces, scarab beetle larvae medicinal materials, silkworm pupa medicinal materials, dead silkworm medicinal materials, and Yixian Pill medicinal materials:

[0103] Take 0.05 g of sample, grind it into powder, put it in a 2 mL centrifuge tube, add 1.2 mL of CTAB precipitate preheated at 56 ° C, mix well, heat in a 65 ° C water bath for 60 min, cool to room temperature, centrifuge at 12000 r / min for 5 min, discard the supernatant, and add 1.0 mL of CTAB precipitate and operate in the same way.

[0104] Add 900 μL CTAB extract, 10 μL proteinase K, and 10 μL β-mercaptoethanol to the centrifuge tube, mix well, and heat in a 65°C water bath for 120 min. Take out the centrifuge tube, cool to room temperature, add an equal volume of chloroform-isoamyl alcohol (24:1), vortex mix well, centrifuge at 12000 r / min, 4°C for 10 min, take the supernatant and put it in a new 2 mL centrifuge tube, and repeat the operation twice.

[0105] Take the supernatant in a 1.5mL centrifuge tube, add an equal volume of isopropanol, and let stand at -20℃ for 60min. Take out the centrifuge tube and centrifuge at 12000r / min for 5min, discard the supernatant, wash the precipitate with 75% ethanol 3 times and anhydrous ethanol once, centrifuge at 12000r / min for 5min, discard the supernatant, incubate the precipitate at 37℃ for 30min, add 30μL of sterile water to dissolve it after the ethanol evaporates, and store it at 4℃ or store it at minus 20℃ for a long time.

[0106] The following method was used to extract genomic DNA from the standard preparation of Bombyx mori and stir-fried Bombyx mori (lyophilized powder), Bombyx mori and stir-fried Bombyx mori formula granule samples:

[0107] Take 0.1 g of sample, grind it into powder, put it in a 2 mL centrifuge tube, add 1.2 mL of CTAB precipitate preheated at 65 ° C, mix well, heat in a 56 ° C water bath for 60 min, cool to room temperature, centrifuge at 12000 r / min for 5 min, discard the supernatant, and add 1.0 mL of CTAB precipitate and operate in the same way.

[0108] Add 900 μL CTAB extract, 10 μL proteinase K, and 10 μL β-mercaptoethanol to the centrifuge tube, mix well, and heat in a 65°C water bath for 120 min. Take out the centrifuge tube, cool to room temperature, add an equal volume of chloroform-isoamyl alcohol (24:1), vortex mix well, centrifuge at 12000 r / min, 4°C for 10 min, take the supernatant and put it in a new 2 mL centrifuge tube, and repeat the operation twice.

[0109] Take the supernatant in a 1.5mL centrifuge tube, add an equal volume of isopropanol, and let stand at -20℃ for 60min. Take out the centrifuge tube and centrifuge at 12000r / min for 5min, discard the supernatant, wash the precipitate with 75% ethanol 3 times and anhydrous ethanol once, centrifuge at 12000r / min for 5min, discard the supernatant, incubate the precipitate at 37℃ for 30min, add 30μL of sterile water to dissolve it after the ethanol evaporates, and store it at 4℃ or store it at minus 20℃ for a long time.

[0110] Subsequently, genomic DNA samples were taken, and the DNA concentration was determined using a BioSpec-nano micro-volume UV spectrophotometer, while OD260 / OD230 and OD260 / OD280 were recorded, and the concentration was adjusted to 100-400 ng / μL.

[0111] S2. Using the genomic DNA as a template, the first primer pair and the second primer pair perform PCR amplification to obtain an amplified product.

[0112] PCR amplification was performed using the first primer pair JiaC07.F / JiaC07.R and the second primer pair BJJ03.F / BJJ03.R to obtain amplified products to verify the applicability of the multiple specific PCR method for Bombyx mori and Beauveria bassiana.

[0113] Establish the multiple specific PCR amplification system and procedure of Bombyx mori and Beauveria bassiana as follows:

[0114] Table 5 PCR amplification (Bombyx mori and Beauveria bassiana) PCR reaction system

[0115]

[0116] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 30 s, for a total of 41 cycles; and finally extension at 72°C for 5 min.

[0117] S3. Performing agarose gel electrophoresis analysis on the amplified product to determine the authenticity of the sample to be tested.

[0118] After the PCR amplification reaction is completed, add 5 μL of 6× loading buffer to the reaction system, mix well, take 6-8 μL of the mixed product and spot it on 1.5% agarose gel, electrophoresed at 150V for 30 minutes, developed with GelRed, and observed the results on a gel imager. Figure 2 .

[0119] according to Figure 2The results showed that the brightness of the target bands of Bombyx mori and Beauveria bassiana was brighter when the primers BJJ03.F / R and JiaC07.F / R were combined and amplified using the 2×Taq PCR premix polymerase system. The bands of Bombyx mori and Beauveria bassiana could be clearly distinguished, and the identification rate was 100%. The identification rate of Bombyx mori and fried Bombyx mori was 100%, the true positive rate was 100%, the true negative rate was 100%, the false positive rate was 0%, and the false negative rate was 0%, indicating that this method can be used for the identification of the authenticity of Bombyx mori and / or fried Bombyx mori and their decoction pieces, water extract products and formula granules, as well as the specific identification of the Chinese patent medicine Yixian Pills mixed with Bombyx mori or fried Bombyx mori powder.

[0120] Example 3

[0121] A method for quickly identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules and its applicability test

[0122] The difference from Example 2 is that in step S1, the samples to be tested are prepared according to the sample list shown in Table 6, and genomic DNA is extracted. The rest is the same as step S1 of Example 2.

[0123] In step S2, the PCR amplification (Bombyx mori and Beauveria bassiana) PCR reaction system shown in Table 7 is used, and the rest is the same as step S2 of Example 2.

[0124] Table 6 Sample list

[0125]

[0126]

[0127] Table 7 PCR amplification (Bombyx mori and Beauveria bassiana) PCR reaction system

[0128]

[0129] PCR amplification was performed using the first primer pair JiaC07.F / JiaC07.R and the second primer pair BJJ03.F / BJJ03.R to obtain amplified products to verify the applicability of the multiple specific PCR method for silkworm and Beauveria bassiana. After the PCR amplification reaction was completed, 5 μL of 6× loading buffer was added to the reaction system and mixed well. 6 to 8 μL of the mixed product was spotted on a 1.5% agarose gel and electrophoresed at 150 V for 30 min. The result was observed on a gel imager with GelRed coloring. The results are shown in the figure. Figure 3 .

[0130] Figure 3The results showed that the target bands of Bombyx mori and Beauveria bassiana obtained by amplification with 2×Pfu PCR premixed polymerase system were brighter, but the bands of some samples were fainter or absent. Among them, the identification of Beauveria bassiana was: the genuine samples 1 to 37, 41, 46 to 47 all had the target bands, and the counterfeit samples 39 to 40, 42 to 45 had no bacterial bands, and the identification rate was 100%; the identification of Bombyx mori was: among the 39 genuine samples, 9 to 10 and 34 had no target bands, and one counterfeit sample (38) had no bands, and the identification rate was 92.5%.

[0131] Compared with the 2×Taq PCR premix polymerase system, the identification rate of the 2×Pfu PCR premix polymerase system is poor, so the 2×Taq PCR premix polymerase system is a system with a higher identification rate.

[0132] The above is a preferred embodiment of the invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention. These improvements and modifications are also considered to be within the scope of protection of the invention.

[0133]

[0134] Sequence Listing <110> Guangdong Yifang Pharmaceutical Co., Ltd. <120> A multiplex specific PCR identification method for extracts of Bombyx batryticatus and fried Bombyx batryticatus and its application in identification of traditional Chinese medicines <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 tctaatatcg cacatagagg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 cagctcatac aaataagggt 20 <210> 3 <211> twenty one <212> DNA <213> Artificial Sequence <400> 3 caacaacgga tctcttggct c 21 <210> 4 <211> twenty three <212> DNA <213> Artificial Sequence <400> 4 ctgtattact acgcagaggt cgc 23

Claims

1. A primer for multiplex PCR identification of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules, characterized in that: comprising a first primer pair and a second primer pair; The sequence of the upstream primer of the first primer pair is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2; The sequence of the upstream primer of the second primer pair is shown in SEQ ID NO:3, and the sequence of the downstream primer thereof is shown in SEQ ID NO:

4.

2. Use of the primer according to claim 1 in (1), (2), (3) or (4): (1) Application in identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules; (2) Application in identifying the authenticity of Chinese patent medicines containing Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, aqueous extracts and granules; (3) Preparation of a kit for identifying Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, aqueous extract products and formulated granules; (4) Application in constructing a method for rapid identification of the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules.

3. The use of the primer according to claim 2 in (1), characterized in that: In the application of identifying the authenticity of silkworm pupa and / or stir-fried silkworm pupa and their decoction pieces, water extract products and formula granules, the application is to simultaneously identify whether silkworm pupa and / or stir-fried silkworm pupa and their decoction pieces, water extract products and formula granules are derived from silkworm and Beauveria bassiana.

4. A kit, characterized in that: It comprises the primer according to claim 1.

5. A method for quickly identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules, characterized in that: The following steps are involved: S1, extracting genomic DNA of the sample to be tested; S2, using the genomic DNA as a template and the primers as claimed in claim 1 to perform PCR amplification to obtain an amplified product; S3. Performing agarose gel electrophoresis analysis on the amplified product to determine the authenticity of the sample to be tested.

6. The method for rapidly identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules as claimed in claim 5, characterized in that: In step S1, the genomic DNA of the sample to be tested is extracted by the following method: Take the sample to be tested, add CTAB precipitation solution for precipitation extraction 1 to 3 times; take the precipitate, add CTAB extraction solution, proteinase K, and β-mercaptoethanol for extraction, and then add chloroform-isoamyl alcohol for extraction 1 to 3 times; take the supernatant after extraction, add isopropanol or isopropanol-sodium acetate for precipitation extraction, wash and incubate the precipitate, and then dissolve it with water to obtain the genomic DNA of the sample to be tested.

7. The method for rapidly identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules as claimed in claim 5, characterized in that: Step S2 includes: The genomic DNA is used as a template and the first primer pair and the second primer pair are used to perform PCR amplification to obtain an amplified product.

8. The method for rapidly identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules as claimed in claim 5, characterized in that: In step S2, the PCR amplification procedure is: pre-denaturing the amplification system at a temperature of 94-96°C for 4-6 minutes, then cycling 38-45 times under a preset program, and finally extending at 71-75°C for 4-6 minutes; The preset program is: first denaturation at 94-96°C for 20-40s, then annealing at 50-56°C for 20-40s, and then extension at 65-80°C for 20-40s.

9. The method for rapidly identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules as claimed in claim 7, characterized in that: The reaction system of the PCR amplification is: 10-15 μL of 2×Taq PCR Mix, 0.3-0.7 μL of the upstream primer of the first primer pair, 0.3-0.7 μL of the downstream primer of the first primer pair, 0.3-0.7 μL of the upstream primer of the second primer pair, 0.3-0.7 μL of the downstream primer of the second primer pair, 0.5-2.5 μL of DNA template, and sterile double distilled water to make up to 25 μL.

10. The method for rapidly identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules as claimed in claim 9, characterized in that: The reaction system for the PCR amplification is: 12.5 μL of 2×Taq PCR Mix, 0.5 μL of the upstream primer of the first primer pair, 0.5 μL of the downstream primer of the first primer pair, 0.5 μL of the upstream primer of the second primer pair, 0.5 μL of the downstream primer of the second primer pair, 1 μL of DNA template, and sterile double distilled water to make up to 25 μL.

11. The method for rapidly identifying the authenticity of Bombyx batryticatus and / or fried Bombyx batryticatus and their decoction pieces, water extract products and formula granules as claimed in claim 5, characterized in that: Step S3 includes: The amplified product is subjected to agarose gel electrophoresis analysis. If the amplified product produces a band at 163 bp, the sample to be tested contains Bombyx mori; if the amplified product produces a band at 260 bp, the sample to be tested contains Beauveria bassiana; When the sample to be tested contains both Bombyx mori and Beauveria bassiana, the sample to be tested is authentic; otherwise, the sample to be tested is a counterfeit product.

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