CN107164471B - Molecular identification method for rapidly identifying truth of beauveria bassiana in traditional Chinese medicine stiff silkworm - Google Patents
Molecular identification method for rapidly identifying truth of beauveria bassiana in traditional Chinese medicine stiff silkworm Download PDFInfo
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Abstract
The invention discloses a primer group for rapidly identifying the authenticity of beauveria bassiana in traditional Chinese medicine stiff silkworm and a molecular identification method. The sequences of the primer groups are respectively shown as SEQ ID NO. 1-2. On the basis of completing high-throughput sequencing of the Guangdong strain beauveria bassiana mitochondrion complete genome sequence and comparative analysis of the mitochondrion complete genome bioinformatics of a plurality of Cordyceps species, the differences of the beauveria bassiana mitochondrion gene sequences on the geographical species are compared, and the ribosomal protein gene of the beauveria bassiana is foundrps3The variation is obvious; 1 pair of primers for specifically detecting the beauveria bassiana are obtained through screening based on the design, a molecular identification method for efficiently and quickly identifying the truth of the beauveria bassiana in the traditional Chinese medicine stiff silkworm is established, the method is simple, convenient and quick, good in specificity, high in sensitivity and wide in applicability, and the truth identification of the beauveria bassiana in the stiff silkworms of different producing areas can be realized; provides technical support for the standardization and normalization of the quality of the traditional Chinese medicine stiff silkworm and the safety of clinical medication.
Description
Technical Field
The invention belongs to the technical field of species identification and Chinese medicinal material authenticity identification. More particularly, relates to a molecular identification method for rapidly identifying the truth of beauveria bassiana in traditional Chinese medicine stiff silkworm.
Background
Bombyx Batryticatus (Bombyx mori), 4-5 th instar larva of Bombyx mori (Bombyx mori L innaeus) of the family Bombycidae, infected (or artificially inoculated) with (or killed by) Beauveria basssaana (Bals.) Vuillant (national pharmacopoeia Committee, 2015), Beauveria bassiana of the family Bombycidae, is a pathogenic fungus parasitic to insects, and it has been reported that NCBI has recently classified the Beauveria bassiana (Dikarya), Ascomycota (Ascomycota), Panomycetes (Pezizomycotina), Chaetomycetozomycetes (Sordariomycetes), Hypocryycetes (Hypocryycetidae), Hypocryycetaleles (Hypocrea), Cordyceps sinensis (Cordypicitaceae), Cordyceps (Cordceps).
The white muscardine silkworm serving as a traditional Chinese medicinal material in China has various pharmacological effects of reducing blood sugar, reducing blood fat, resisting cancer, resisting convulsion, resisting coagulation, inhibiting bacteria, tranquilizing, hypnotizing, whitening and the like, so that the white muscardine silkworm has an important effect on clinical treatment. At present, the income brought by the silkworm breeding of farmers is considerable, and the silkworm breeding method can be used as a good way for farmers to become rich (Zhang Wei, 2016). However, some merchants have begun to use counterfeit and non-qualified stiff silkworms as qualified stiff silkworms for greater benefit due to their important pharmacological effects and good economic benefits. At present, most of the traditional counterfeit silkworm bombyx batryticatus is processed by mixing white silkworm with lime water (Zhao, 1998), and also the wild silkworm is used as the certified silkworm (Hu Da ze, et al, 2003), and in recent years, new counterfeit black dead silkworm and hollow silkworm have appeared. Because the appearance, the microscopic appearance and the like of the counterfeit stiff silkworm are similar to those of the certified stiff silkworm, the counterfeit stiff silkworm is difficult to effectively identify by using a conventional method, and the authenticity identification of the counterfeit stiff silkworm is particularly important in the quality evaluation work of the white stiff silkworm in order to avoid the phenomenon.
At present, the existing batryticated silkworm quality identification methods mainly comprise two methods, one is a physical identification method, which comprises the methods of appearance identification and microscopic observation (Xuzhou equalling, 1996; national pharmacopoeia committee, 2015), micro sublimation method (yellow dawn snow, 2005), SDS-PAGE (Qiu's B, 2014), grey correlation degree analysis method (plum peak, 2011), ultraviolet fingerprint (Ji's neck, 2006), infrared fingerprint (Ji's neck, 2007), high performance liquid chromatography (Wang Xian, 2006) and the like to identify the medicinal material quality of the bated silkworm; and secondly, a molecular identification method, which is mainly used for respectively identifying silkworm and beauveria bassiana which are basic species of the medicinal material of the stiff silkworm based on a COI sequence and an ITS sequence, such as Jiajing (2016), so as to identify the quality and the quality of the white stiff silkworm. The molecular identification has the characteristics of more accuracy, simple and convenient operation and the like, and has better application prospect.
At present, in the aspect of identifying the truth of the beauveria bassiana in the stiff silkworms, primers for specifically identifying the beauveria bassiana are always lacked, and a molecular identification method for rapidly identifying the truth of the beauveria bassiana in the traditional Chinese medicinal material stiff silkworms is lacked.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects and shortcomings of the existing bombyx batryticatus authenticity identification technology, completing high-throughput sequencing of the Guangdong strain beauveria bassiana mitochondrion whole genome sequence, comparing the difference of beauveria bassiana mitochondrion gene sequences in geographical species, and finding that the variation of the beauveria bassiana ribosomal protein genes rps3 is obvious; and designing a primer based on a muscardine silkworm mitochondrial rps3 gene sequence, searching a primer for specifically amplifying the muscardine fungi, and providing a molecular marking method for specifically identifying the muscardine fungi, so that the authenticity of the muscardine fungi in the stiff silkworms can be quickly identified, and a molecular technical support is provided for the safe work of clinical medication of the traditional Chinese medicinal material stiff silkworms.
The invention aims to provide the sequence of ribosomal protein gene rps3 of beauveria bassiana.
The invention also aims to provide a primer group for identifying and verifying the truth of the beauveria bassiana in the stiff silkworms.
The invention further aims to provide a molecular identification method for rapidly identifying the truth of the beauveria bassiana in the traditional Chinese medicine stiff silkworm.
The invention further aims to provide a kit for rapidly identifying the truth of the beauveria bassiana in the traditional Chinese medicine stiff silkworm.
The above purpose of the invention is realized by the following technical scheme:
the inventor discovers that the ribosomal protein gene rps3 of the beauveria bassiana has obvious variation on the basis of completing sequencing of the whole mitochondrial genome of the beauveria bassiana in Guangdong and comparative analysis of the whole mitochondrial genome of 13 cordyceps species in bioinformatics, and designs and screens 1 pair of primers for specifically detecting the beauveria bassiana on the basis of the rps3 gene sequence of the beauveria bassiana in Guangdong.
A group of primers for identifying and verifying the authenticity of beauveria bassiana in the stiff silkworms comprises a primer pair F935 and a primer pair R1385, and the sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
The application of the primer group in identifying and verifying the truth of the beauveria bassiana in the stiff silkworms, the application in constructing a molecular identification method for quickly identifying the truth of the beauveria bassiana in the traditional Chinese medicinal material stiff silkworms and the application in preparing a kit for quickly identifying the truth of the beauveria bassiana in the traditional Chinese medicinal material stiff silkworms are all within the protection scope of the invention.
A molecular identification method for rapidly identifying the truth of white muscardine fungi in traditional Chinese medicine silkworm comprises the steps of taking the total DNA of the traditional Chinese medicine silkworm as a template, utilizing a primer group F935/R1385 to carry out PCR amplification, carrying out agarose gel electrophoresis on an amplification product, and judging whether the sample of the traditional Chinese medicine silkworm contains the white muscardine fungi according to the existence of a clear electrophoresis strip of 500bp in the PCR amplification result.
Further preferably, sequencing and data comparison can be continuously carried out on the PCR amplification product, a phylogenetic tree is constructed, and the result is further determined.
Preferably, the extraction method of the total DNA of the silkworm larva comprises the following steps: pretreating white muscardine silkworm into powder, and extracting total DNA; the pretreatment is as follows: wiping the surface of the silkworm larva with 65-75% ethanol, putting the silkworm larva in an environment with the temperature of 30-40 ℃ to volatilize the ethanol, and then crushing and grinding the silkworm larva into powder.
More preferably, the extraction method of the total DNA of the bombyx batryticatus comprises the following steps: wiping the surface of Bombyx Batryticatus with 70% ethanol, volatilizing ethanol at 37 deg.C, and pulverizing with high-speed multifunctional pulverizer; then adding liquid nitrogen for full grinding, using a fungus genome rapid extraction kit to extract total DNA, and storing at-20 ℃ for later use.
In addition, the reaction system of the PCR is preferably 2xTaq Master Mix 25. mu.l, 10 pmol/. mu. L upstream and downstream primers 1. mu.l each, DNA template 1. mu.l, ddH2O 2μl。
Preferably, the reaction conditions of the PCR are: 5min at 94 ℃; 30s at 94 ℃, 30s at 44 ℃, 45s at 72 ℃ and 25 cycles; 10min at 72 ℃.
The invention also provides a kit containing the primer pair F935 and R1385 for rapidly identifying the truth of the beauveria bassiana in the traditional Chinese medicine stiff silkworm, and the kit is also within the protection scope of the invention.
Preferably, the kit can also comprise reagents required for PCR amplification by using the total DNA of the Chinese medicinal material silkworm larva to be detected as a template and using a primer group F935/R1385.
The application method of the kit is the molecular identification method for rapidly identifying the truth of the beauveria bassiana in the traditional Chinese medicine stiff silkworm.
On the basis of completing sequencing of the Guangdong strain beauveria bassiana mitochondria whole genome and comparative analysis of 13 cordyceps family mitochondria whole genome bioinformatics, the invention discovers that the variation of the ribosomal protein gene rps3 of the beauveria bassiana is obvious, designs a synthetic primer based on the Guangdong strain beauveria bassiana mitochondria whole gene sequence, and finally screens 1 pair of primers for specifically detecting the beauveria bassiana. And the primer validity verification is carried out by utilizing 12 geographical strain beauveria bassiana and a commercial stiff silkworm sample, the verification of the specificity and the validity of the designed primer is completed, and the molecular identification method for efficiently and quickly identifying the truth of the beauveria bassiana in the traditional Chinese medicine stiff silkworm is established.
The invention has the following beneficial effects:
based on the comparison and analysis of the Guangdong beauveria bassiana mitochondria whole genome sequencing result and 13 cordyceps family mitochondria whole genome bioinformatics, and the multi-sequence comparison and analysis of 12 different geographical strain beauveria bassiana rps3 genes, the invention discovers that partial sites of rps3 sequences of different geographical strain beauveria bassiana have base substitution phenomenon, so that the gene rps3 is selected as a molecular marker of the beauveria bassiana mitochondria whole genome to be applied to the identification of the beauveria bassiana in the traditional Chinese medicinal material of the stiff silkworms, and provides a theoretical basis for the identification of the truth of the stiff silkworms.
Meanwhile, a high-throughput sequencing library is constructed by extracting the total genomic DNA of the Guangdong strain beauveria bassiana, and sequencing is carried out by adopting a lluminaHiseq2500 platform; the method of denovo assembles the sequencing data into complete beauveria mitochondrial genome sequences. 1 pair of primers for specifically detecting the beauveria bassiana are designed and screened based on the Guangdong strain beauveria bassiana mitochondrion complete gene sequence, the specificity is good, the sensitivity is high, and therefore a new molecular identification method is provided for identifying the authenticity of the beauveria bassiana in the commercial batryticated silkworms in different producing areas.
The primer pair for specifically identifying the beauveria bassiana designed by the invention is used for identifying the truth of the beauveria bassiana in commercially available batryticated silkworms in different producing areas, and finally, a phylogenetic tree is constructed through PCR amplification, electrophoresis, sequencing and data comparison, so that experimental reference and theoretical basis are provided for molecular identification of the beauveria bassiana in the batryticated silkworms, and a new method is provided for quality standardization and normalization of the traditional Chinese medicinal material of the batryticated silkworms.
Drawings
FIG. 1 shows the alignment of the gene sequences of 12 geographical strains of Beauveria bassiana rps 3.
FIG. 2 shows the alignment of the gene sequences of 12 geographical strains of Beauveria bassiana rps 3.
FIG. 3 shows the alignment of the gene sequences of 12 geographical strains of Beauveria bassiana rps 3.
FIG. 4 is a diagram showing the structure of mitochondrial genes of beauveria bassiana of the Guangdong strain.
FIG. 5 is a phylogenetic tree constructed by the Guangdong strain Beauveria bassiana near-source species.
FIG. 6 shows the PCR amplification result of a primer set F935/R1385 designed based on the mitochondrial rps3 gene of beauveria guangdongensis, wherein M is D L2000 DNA Marker, 1: laboratory-preserved strain Beauveria bassiana B.basiana, 2: Guangdong-English strain Beauveria bassiana B.basiana, 3: Guangxi-Nanning strain Beauveria bassiana B.basiana, 4: Guangdong-English strain Beauveria serotype B.basiana (Serum _ types), 5: Guangdong-English strain Isaria japonica, 6: Aspergillus oryzae Nomia, 7: Fusarium proliferatum, 8: water blank control group.
FIG. 7 shows the PCR amplification results of 12 geostrain Beauveria bassiana based on the primer set F935/R1385. Note: M: Marker 2000D L, 1: laboratory preserved strain, 2: Guangxi Zhongping strain, 3: Guangxi Zhongpinglanguancun strain, 4: Guangdong MaoMing strain, 5: Guangdong Huazhou strain, 6: Guangxi Yizhou Shibie strain, 7: Guangxi Huanjiang strain, 8: Guangdong Wenyuan Nippon strain, 9: Guangxi Nanning strain, 10: Guangdong institute for microbiology biocontrol strain, 11: Guangdong English strain, 12: Hunan Luxi strain, and 13: water blank set.
FIG. 8 is a phylogenetic tree constructed based on 12 geographical strains of Beauveria bassiana rps3 gene.
FIG. 9 shows the PCR amplification results of primers F935/R1385 on Bombyx Batryticatus of different origins, wherein M is Marker 2000D L, No. 1-41 are commercially available Bombyx Batryticatus of different origins, and No. A, B is water blank.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 Beauveria bassiana specific primer design
1. Based on a mitochondrial complete genomics analysis method, the inventor compares the mitochondrial complete genome sequence gene structures of 13 cordyceps species including beauveria bassiana, finds 1 beauveria bassiana differential protein coding gene rps3, wherein the gene is a gene for coding ribosomal protein S3, and the ribosomal protein S3 is a constituent protein of ribosomal 40S small subunit, has the ribose in vitro function and plays an important role in the processes of DNA damage repair, gene expression regulation, apoptosis and the like.
Meanwhile, C L USTA L X1.81 software is used for carrying out multi-sequence comparison analysis on 12 different geographical strain beauveria bassiana rps3 genes, and the result shows that a base substitution phenomenon exists at a part of the sites of the rps3 sequences of the 12 different geographical strain beauveria bassiana (the comparison result of the 12 geographical strain beauveria bassiana rps3 gene sequences is shown in figures 1, 2 and 3, and the figures 1, 2 and 3 are sequentially continuous).
Therefore, the gene rps3 is selected as a molecular marker of the whole mitochondrial genome of the beauveria bassiana and is applied to the authenticity identification of the beauveria bassiana in the traditional Chinese medicine of the stiff silkworm.
2. Sequencing the mitochondrial whole genome of Guangdong strain beauveria bassiana
The structure diagram of the mitochondrial gene of beauveria bassiana of Guangdong strain is shown in FIG. 4. The mitochondria of the Guangdong strain beauveria bassiana is of a closed ring structure, the total length of a basic group is 29922bp, the GC proportion is 27.26 percent, 42 coding genes (15 protein coding genes, 25 tRNA and 2 rRNA) are contained, and the GAP region is not contained.
The specific method comprises the following steps:
s1, propagation, separation and purification of Guangdong strain beauveria bassiana strain
Preparing a PDA culture medium, and propagating, separating and purifying the Guangdong strain beauveria bassiana strain by adopting a scribing method; preparing single bacterial colony of Guangdong strain beauveria bassiana.
S2, extracting the total DNA of the Guangdong strain beauveria bassiana genome
Taking a proper amount of single colony hypha of beauveria bassiana, adding liquid nitrogen, fully grinding, and extracting total DNA of diseased leaves by using a Kangji fungus DNA extraction kit according to the operation instruction.
S3, constructing a high-throughput sequencing library
S4.Illumina Hiseq2500 platform sequencing
S5, quality detection
In order to ensure the reliability of subsequent analysis, necessary detection and data screening are carried out on sequencing data, and the detection content comprises the following steps: data quality detection, sequencing data quantity, sequencing data quality, GC proportion distribution, sequencing accuracy and the like; and simultaneously, screening the data based on the detection result, wherein the screening standard is as follows: the sequencing data Q20 (the percentage of bases with accuracy of 99% or more) was not less than 90%, and Q30 (the percentage of bases with accuracy of 99.9% or more) was not less than 83%.
S6, mitochondrial genome sequence capture
And (3) according to the published mitochondrial genome sequence of the kindred object, separating the mitochondrial genome sequence of the sequencing sample from the total DNA sequencing data based on a DNA sequence comparison method.
S7, genome assembly and verification
And assembling the sequenced DNA sequence fragments into a complete mitochondrial genome according to the overlap relation of the mitochondrial genome data. And the correctness of the assembly is verified by the sequencing quality and the sequencing depth.
S8 genome annotation
The annotation of the genome starts from tRNA, and the tRNA is annotated by MiTFi, tRNAscan-SE (V1.21) and ARWEN to ensure the accuracy of tRNA annotation; the annotation of coding gene and rRNA gene is carried out together based on the method of blastn & blastx (2.2.28+), after the specific gene sequence is determined by comparing the results, the specific gene sequence is translated into protein sequence and annotated to verify whether the genome annotation is correct.
S9. evolution analysis
A systematic evolution tree is constructed by adopting a RAxM L (V8.1.5) software maximum likelihood method, the Bootstrap value is 1000, the adopted optimal nucleotide model is GTR + G + I, and the optimal nucleotide model is CpREV + I + G + F.
S10, biological information analysis result summarization
The beauveria bassiana mitochondrial genome adopts an Illumina Hiseq2500 platform to perform high-throughput sequencing, a PE sequencing library is constructed, and sequencing data are finally summarized.
Further, a phylogenetic tree was constructed according to sequencing and data comparison, and as shown in fig. 5, the species classification of beauveria bassiana, guangdong, was determined.
3. Primer design
Based on the completed high-throughput sequencing result of the mitochondrial rps3 gene of the beauveria bassiana strain in Guangdong, a plurality of groups of primers are designed, and 6 pairs of primer groups shown in the table 1 are determined for further experiments through preliminary screening.
TABLE 1 primer design and sequence information based on Beauveria bassiana mitochondrial rps3 gene
4. Primer validation
(1) The PCR amplification system and procedure were set up as follows:
TABLE 2 PCR reaction System (50. mu. L System)
And (3) PCR reaction conditions: 5min at 94 ℃; 30s at 94 ℃, 30s at 44 ℃, 45s at 72 ℃ and 25 cycles; 10min at 72 ℃.
(2) PCR amplification
The primer group shown in Table 1 was used for PCR amplification, and the product was subjected to agarose gel electrophoresis, and the results showed that the primer group I, i.e., the primer group F935/R1385, was capable of specifically detecting Beauveria bassiana.
The PCR amplification result of the primer group F935/R1385 is shown in FIG. 6, and the lane 8 (water blank control) has no reaction band, which indicates that the PCR reaction process has no pollution phenomenon and reliable reaction result, and the PCR amplification result obtains an electrophoresis band with the target fragment size of about 500 bp. The primer group F935/R1385 designed based on the Guangdong strain beauveria bassiana mitochondrial rps3 gene can amplify beauveria bassiana ( lanes 1, 2 and 3) and beauveria bassiana serotype (lane 4) but can not amplify the fungi such as beauveria bassiana (lane 5), aspergillus (lane 6) and fusarium (lane 7), and the primer group F935/R1385 can be used as a specific primer for rapidly identifying the beauveria bassiana.
Example 212 PCR amplification of the Gene of the geographical strain Beauveria bassiana rps3
1. The DNA of 12 geographical strains of beauveria bassiana is used as a template, and a primer group F935/R1385 is used for PCR amplification to further verify the effectiveness of the primers.
2. The results are shown in FIG. 7, and no reaction band appears in lane 13 (water blank), indicating that there is no contamination during the PCR reaction and the reaction results are reliable. Clear electrophoresis bands are obtained from the PCR amplification results of 12 geographical strain beauveria bassiana, and the sizes of target fragments are about 500 bp. The verification of the effectiveness of the primers is completed by the graph shown in FIG. 7, and the primer group F935/R1385 can be used as a specific primer for identifying beauveria bassiana.
3. Furthermore, the amplified product can be continuously sequenced and compared with data, a phylogenetic tree is constructed, and the result is further determined. The phylogenetic tree constructed based on 12 geographical strain beauveria bassiana rps3 genes is shown in fig. 8.
Example 3 identification of Beauveria bassiana in commercial Bombyx Batryticatus samples based on primer set F935/R1385
1. Experimental methods
(1) Commercial Bombyx Batryticatus total DNA extraction
Sample pretreatment, wiping the surface of the silkworm larva with 70% ethanol, placing the silkworm larva in a glass dish, volatilizing the ethanol in a drying oven at 37 ℃, grinding the silkworm larva into powder by using a high-speed multifunctional grinder, and filling the powder into a 25m L sterilized plastic tube for later use.
Taking a proper amount of stiff silkworm powder, adding liquid nitrogen, fully grinding, extracting the total DNA of the beauveria bassiana mycelium by using a fungal genome rapid extraction kit purchased from Shanghai biological engineering Co., Ltd, and storing at-20 ℃ for later use.
(2) Identification of beauveria bassiana in commercial batryticated silkworm in different producing areas
The whole genome DNA of the traditional Chinese medicine stiff silkworm is used as a template, and a primer group F935/R1385 is used for PCR amplification.
2. The PCR amplification results are shown in FIG. 9, and it can be seen from the PCR amplification results of the different producing areas of Bombyx Batryticatus by the primer group F935/R1385 that whether the silkworm samples in the market producing areas contain Beauveria bassiana can be judged according to the clear electrophoresis band with 500bp mesh, thereby identifying the truth of Beauveria bassiana in the commercial Bombyx Batryticatus in the different producing areas.
3. Furthermore, the amplified product can be subjected to sequencing and data comparison continuously, a phylogenetic tree is constructed, the result is further determined, and a basis is provided for a molecular identification method for identifying the truth of the beauveria bassiana in the bombyx batryticatus.
SEQUENCE LISTING
<110> southern China university of agriculture
<120> molecular identification method for rapidly identifying authenticity of beauveria bassiana in traditional Chinese medicine stiff silkworm
<130>
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<170>PatentIn version 3.3
<210>1
<211>18
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<213> primer F935
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Claims (10)
1. A group of primers for identifying and verifying the authenticity of beauveria bassiana in the stiff silkworms is characterized in that the primers are a primer pair F935 and a primer pair R1385, and the sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
2. The use of the primer set according to claim 1 for identifying and verifying the authenticity of beauveria bassiana in bombyx batryticatus.
3. The use of the primer set of claim 1 in the construction of a molecular identification method for rapidly identifying the authenticity of beauveria bassiana in a traditional Chinese medicinal material of Bombyx Batryticatus.
4. The use of the primer set of claim 1 in the preparation of a kit for rapidly identifying the authenticity of beauveria bassiana in a traditional Chinese medicinal material of Bombyx Batryticatus.
5. A molecular identification method for rapidly identifying the truth of white muscardine fungi in traditional Chinese medicine silkworm is characterized in that total DNA of the traditional Chinese medicine silkworm to be detected is used as a template, a primer group F935/R1385 in claim 1 is used for PCR amplification, an amplification product is subjected to agarose gel electrophoresis, and whether the white muscardine fungi are contained in a traditional Chinese medicine silkworm sample to be detected or not is judged according to the clear 500bp electrophoresis strip of a PCR amplification result.
6. The molecular identification method of claim 5, wherein the extraction method of total DNA of Bombyx Batryticatus comprises: pretreating white muscardine silkworm into powder, and extracting total DNA; the pretreatment is as follows: wiping the surface of the silkworm larva with 65-75% ethanol, putting the silkworm larva in an environment with the temperature of 30-40 ℃ to volatilize the ethanol, and then crushing and grinding the silkworm larva into powder.
7. The molecular characterization method of claim 5 wherein the PCR reaction system is 2xTaqMaster Mix 25. mu.l, 10 pmol/. mu. L upstream and downstream primers 1. mu.l, DNA template 1. mu.l, ddH2O 2μl；
The reaction conditions of the PCR are as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 44 ℃, 45s at 72 ℃ and 25 cycles; 10min at 72 ℃.
8. The molecular characterization method of claim 5, further comprising sequencing the PCR amplification product and comparing the data to construct a phylogenetic tree to further determine the result.
9. A kit for rapidly identifying the authenticity of beauveria bassiana in traditional Chinese medicine stiff silkworm is characterized by comprising the primer pair F935 and R1385 in claim 1.
10. The kit of claim 9, further comprising reagents required for performing PCR amplification by using a primer set F935/R1385 with total DNA of Bombyx Batryticatus as a template.
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| CN107937567B (en) * | 2017-12-29 | 2020-09-25 | 江苏大学 | Specific primers for identifying bombyx batryticatus and identification method thereof |
| CN112730291B (en) * | 2020-12-23 | 2022-09-30 | 江苏省中医院 | Qualitative and quantitative marker of silkworm counterfeit product and detection method thereof |
| CN114113376B (en) * | 2021-11-10 | 2023-08-29 | 广东一方制药有限公司 | Method for identifying characteristic polypeptide of stiff silkworm, water extract product of stiff silkworm and other stiff silkworm products |
| CN116068096B (en) * | 2023-04-04 | 2023-06-16 | 成都中医药大学 | Specific marker related to stiff silkworm section silk gland ring, screening method and stiff silkworm quality detection method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7241612B2 (en) * | 2002-08-20 | 2007-07-10 | The United States Of America, As Represented By The Secretary Of Agriculture | Methods and materials for control of insects such as pecan weevils |
| CN106048020A (en) * | 2016-06-15 | 2016-10-26 | 中国医学科学院药用植物研究所 | Gene identification card for animal medicine stiff silkworm |
-
2017
- 2017-05-18 CN CN201710354083.8A patent/CN107164471B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7241612B2 (en) * | 2002-08-20 | 2007-07-10 | The United States Of America, As Represented By The Secretary Of Agriculture | Methods and materials for control of insects such as pecan weevils |
| CN106048020A (en) * | 2016-06-15 | 2016-10-26 | 中国医学科学院药用植物研究所 | Gene identification card for animal medicine stiff silkworm |
Non-Patent Citations (3)
| Title |
|---|
| accession: NC_010652.2，"Beauveria bassiana mitochondrion, complete genome";无;《Genbank》;20081209;第1-2页 * |
| Molecular Evolution of the mtDNA Encoded rps3 Gene Among Filamentous Ascomycetes Fungi with an Emphasis on the Ophiostomatoid Fungi;Jyothi Sethuraman等;《J Mol Evol》;20091014;第69卷;摘要，第372页左栏最后1段至第382页右栏最后1段 * |
| Sequence and phylogenetic analysis of Beauveria;XU Jin-Zhu等;《Mycosystema》;20090915;第28卷(第5期);第718-723页 * |
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