CN107164471A - The method for identifying molecules of the muscardine true and false in a kind of quick discriminating Chinese medicine stiff silkworm - Google Patents
The method for identifying molecules of the muscardine true and false in a kind of quick discriminating Chinese medicine stiff silkworm Download PDFInfo
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Abstract
The invention discloses the primer sets of the muscardine true and false in one group of quick discriminating Chinese medicine stiff silkworm and method for identifying molecules.The primer sets sequence is respectively as shown in SEQ ID NO.1~2.The present invention is on the basis of the mitochondria full-length genome bioinformatics comparative analysis to the high-flux sequence and multiple cordyceps sinensis sections species of Guangdong Strain muscardine mitochondria whole genome sequence is completed, compare difference of the muscardine Mitochondrial gene sequence in geographical kind, find the ribosomal protein gene of muscardinerps3Variation is obvious；And the primer of 1 pair of energy specific detection muscardine has been obtained based on this design screening, establish the method for identifying molecules of the muscardine true and false in efficient, quick discriminating Chinese medicine stiff silkworm, the easy quick, specificity of method is good, sensitivity is high, and applicability is wide, the discriminating to the muscardine true and false in different sources stiff silkworm can be achieved；Quality standardization and standardization and clinical application for Chinese medicine stiff silkworm have safely provided technical support.
Description
Technical field
The invention belongs to species identification and Chinese medicine true and false authentication technique field.More particularly, to a kind of quick discriminating
The method for identifying molecules of the muscardine true and false in Chinese medicine stiff silkworm.
Background technology
The larva of a silkworm with batrytis (Bombyx mori), the larva in Bombycidae insect silkworm (Bombyx mori Linnaeus) 4~5 ages
Infect (or artificial infection) muscardine beauveria bassiana (Bals.) Vuillant and lethal hirudo leech (national medicine
The allusion quotation committee, 2015).Muscardine in the larva of a silkworm with batrytis is the disease fungus that a class parasitizes insect, according to NCBI latest datas,
Muscardine is ranged：Double-core suberathem (Dikarya), Ascomycota (Ascomycota), cup fungi subphylum
(Pezizomycotina), excrement shell Gammaproteobacteria (Sordariomycetes), meat seat bacterium subclass (Hypocreonycetidae), meat seat
Zoopagales (Hypocreales), cordyceps sinensis Cordycepps (Cordycipitaceae), Cordyceps (Cordceps).
The larva of a silkworm with batrytis is as the traditional Chinese medicine of China, with hypoglycemic, reducing blood lipid, anticancer, anticonvulsion, anti-freezing, antibacterial, town
The multiple pharmacological effects such as quiet hypnosis, whitening, so that it has important effect on clinical treatment.Current peasant household's cultivation is stiff
The income that silkworm is brought be it is very considerable, can as farmer richness good approach (Zhang Weiji, 2016).However, due to stiff silkworm
With important pharmacological action and good economic benefit so that some businessmans in order to obtain more interests, start with personation and
Unqualified stiff silkworm serves as qualified stiff silkworm.Traditional adulterant stiff silkworm is mostly that dead silkworm is mixed into white process with limewash at present
(Zhao Qiyou, 1998), also has and pretends to be certified products stiff silkworm to use (Hu great Ze etc., 2003) cutworm, in recent years, occur in that new adulterant
Black dead silkworm and hollow stiff silkworm.Due to the outward appearance, micro- etc. similar to certified products of adulterant stiff silkworm, it is difficult to had using conventional method
Effect differentiates, in order to prevent the appearance of such phenomenon, and its true and false differentiates to be particularly important in larva of a silkworm with batrytis performance rating work.
The stiff silkworm Quality evaluation of present mainly includes two kinds, and one is physical characterization method, including appearance identification
With microexamination (Xu state equalization, 1996；Chinese Pharmacopoeia Commission, 2015), microsublimation method (Huang Xiaoxue, 2005), SDS-
PAGE (Qiu Yi, 2014), grey Relational Analysis Method (Li Feng, 2011), ultraviolet fingerprint (Ji Xianling, 2006), infrared finger
The methods such as line collection of illustrative plates (Ji Xianling, 2007), high performance liquid chromatography (Wang Gengxian, 2006) are identified stiff silkworm quality of medicinal material；
Two be method for identifying molecules, the base thing based on COI sequences and ITS sequence respectively to stiff silkworm medicinal material such as mainly Jia Jing (2016)
Plant silkworm and muscardine is identified, so that the quality and quality to the larva of a silkworm with batrytis are identified.Wherein, Molecular Identification has more accurate
Really, easy to operate the features such as, application prospect is more preferable.
At present to muscardine authenticity aspect in stiff silkworm, the primer of special checking muscardine is lacked always, lacks a kind of
The method for identifying molecules of the muscardine true and false in quick discriminating Chinese medicine stiff silkworm.
The content of the invention
The technical problem to be solved in the present invention is the defect and deficiency for overcoming existing stiff silkworm authenticity technology, is completed to wide
The high-flux sequence of eastern strain muscardine mitochondria whole genome sequence, compares muscardine Mitochondrial gene sequence in geographical kind
Difference, it is found that the ribosomal protein gene rps3 variations of muscardine are obvious；And based on muscardine mitochondria rps3 gene orders
Primer is designed, the primer of specific amplified fungi muscardine is found there is provided a kind of molecule labelling method of unique identification muscardine, from
And in quick discriminating stiff silkworm muscardine the true and false, be on Chinese medicine stiff silkworm clinical application trouble free service provide molecular engineering support.
It is an object of the invention to provide the ribosomal protein gene rps3 sequences of muscardine.
Another object of the present invention is to provide the primer sets of the muscardine true and false in one group of identification checking stiff silkworm.
Still a further object of the present invention is to provide a kind of method for identifying molecules of the muscardine true and false in quick discriminating Chinese medicine stiff silkworm.
Another object of the present invention is to provide a kind of kit of the muscardine true and false in quick discriminating Chinese medicine stiff silkworm.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
The present inventor completes the line grain to Guangdong Strain muscardine mitochondria genome sequencing and 13 cordyceps sinensis section species
On the basis of body full-length genome bioinformatics comparative analysis, it is found that the ribosomal protein gene rps3 variations of muscardine are obvious,
And based on the design screening of Guangdong Strain muscardine mitochondrial rps3 gene orders obtained 1 to can specific detection muscardine draw
Thing.
The primer sets of the muscardine true and false in one group of identification checking stiff silkworm, are primer pair F935 and R1385, and sequence is respectively such as
Shown in SEQ ID NO.1 and SEQ ID NO.2.
Application of the primer sets in identification checking stiff silkworm in the muscardine true and false, is building quick discriminating Chinese medicine stiff silkworm
Application in the method for identifying molecules of the middle muscardine true and false, and the muscardine true and false in quick discriminating Chinese medicine stiff silkworm is prepared
Application in kit, all should be within protection scope of the present invention.
The method for identifying molecules of the muscardine true and false in a kind of quick discriminating Chinese medicine stiff silkworm, it is total with the Chinese medicine larva of a silkworm with batrytis to be measured
DNA is template, enters performing PCR amplification using primer sets F935/R1385, amplified production enters row agarose gel electrophoresis, according to PCR
Whether the electrophoresis strip that amplification whether there is clearly 500bp mesh brings and judges in Chinese medicine larva of a silkworm with batrytis sample to be measured containing white stiff
Bacterium.
It is further preferred that can also continue to that pcr amplification product is sequenced and data comparison, constructing system chadogram,
Further determine that result.
Wherein it is preferred to, the extracting method of the larva of a silkworm with batrytis STb gene is：First the larva of a silkworm with batrytis is pre-processed into after powder, carried out
Genome DNA extraction；The pretreatment refers to：Behind 65~75% ethanol larva of a silkworm with batrytis surfaces, being put in 30~40 DEG C of environment makes
Alcohol volatilizees, then crushes milling.
Specifically it is highly preferred that the extracting method of the larva of a silkworm with batrytis STb gene is：First with 70% ethanol larva of a silkworm with batrytis surface
Afterwards, being put in 37 DEG C of environment makes alcohol volatilize, then is milled with high-speed multifunctional pulverizer；Then add liquid nitrogen to be fully ground, make
STb gene is extracted with fungal gene group rapid extraction kit, -20 DEG C is placed in and saves backup.
Furthermore it is preferred that the reaction system of the PCR is：Above and below μ l, the 10pmol/ μ L of 2xTaq Master Mix 25
Swim each 1 μ l of primer, DNA profiling 1 μ l, ddH2O 2μl。
Preferably, the reaction condition of the PCR：94℃5min；94 DEG C of 30s, 44 DEG C of 30s, 72 DEG C of 45s, 25 circulations；72
℃10min。
The examination of the muscardine true and false in a kind of quick discriminating Chinese medicine stiff silkworm for including above-mentioned primer pair F935 and R1385
Agent box, also within protection scope of the present invention.
Preferably, the kit can also include using Chinese medicine larva of a silkworm with batrytis STb gene to be measured as template, utilize primer sets
The reagent that F935/R1385 enters needed for performing PCR amplification.
The Molecular Identification side of the muscardine true and false in the application method quick discriminating Chinese medicine stiff silkworm as described above of the kit
Method.
The present invention is completed to Guangdong Strain muscardine mitochondria genome sequencing and the mitochondria of 13 cordyceps sinensis section species
On the basis of full-length genome bioinformatics comparative analysis, it is found that the ribosomal protein gene rps3 variations of muscardine are obvious, and
Synthetic primer is designed based on Guangdong Strain muscardine mitochondria complete genome sequence, finishing screen have selected 1 pair can specific detection muscardine
Primer.And primer validation verification is carried out using 12 plants of geographical strain muscardines and commercially available stiff silkworm sample, complete and pair set
Primer specificity, the checking of validity are counted, the Molecular Identification of the muscardine true and false in efficient, quick discriminating Chinese medicine stiff silkworm is established
Method.
The invention has the advantages that：
The present invention is based on the line grain to Guangdong Strain muscardine mitochondria genome sequencing result and 13 cordyceps sinensis section species
Body full-length genome bioinformatics comparative analysis, and multisequencing pair is carried out to 12 plants of strains system muscardine rps3 genes
On the basis of analysis, it is found that the moiety site of the rps3 sequences of strains system muscardine has the replacement phenomenon of base,
Therefore Select gene rps3 is true applied to muscardine in Chinese medicine stiff silkworm as the molecular labeling of muscardine mitochondria full-length genome
Puppet differentiates, theoretical foundation is provided for the true and false discriminating of stiff silkworm.
Meanwhile, by extracting Guangdong Strain muscardine genome DNA, high-throughput sequencing library is built, using llumina
Hiseq2500 platforms are sequenced；Sequencing data is assembled into complete muscardine mitochondrial genomes sequence by denovo method
Row.The primer for having filtered out 1 pair of energy specific detection muscardine is designed based on Guangdong Strain muscardine mitochondria complete genome sequence, specifically
Property good, sensitivity it is high, so as to be the new Molecular Identification side of offer that the base muscardine true and false differentiates in the commercially available stiff silkworm of different sources
Method.
Muscardine is true in this commercially available stiff silkworm of primer pair different sources to unique identification muscardine designed using the present invention
Puppet differentiated, expanded eventually through PCR, electrophoresis, sequencing and data comparison, constructing system chadogram, be muscardine in stiff silkworm
Molecular Identification provide laboratory reference and theoretical foundation, the quality standardization and standardization for Chinese medicine stiff silkworm provide new square
Method.
Brief description of the drawings
Fig. 1 is 12 plants of geographical strain muscardine rps3 gene order comparison results.
Fig. 2 is 12 plants of geographical strain muscardine rps3 gene order comparison results.
Fig. 3 is 12 plants of geographical strain muscardine rps3 gene order comparison results.
Fig. 4 is Guangdong Strain muscardine chondriogen structure chart.
Fig. 5 is the systematic evolution tree constructed by the nearly source species of Guangdong Strain muscardine.
The PCR that Fig. 6 is the primer sets F935/R1385 designed based on Guangdong Strain muscardine mitochondria rps3 genes expands knot
Really.Note：M is DL2000 DNA Marker；1：Laboratory preservation strain muscardine B.bassiana, 2：Yingde, Guangdong strain muscardine
B.bassiana, 3：Nanning strain muscardine B.bassiana, 4：Yingde, Guangdong strain muscardine serotypes B .bassiana
(Serum_types), 5：Yingde, Guangdong strain ash deadlock bacterium Isaria javanica, 6：Aspergillus Aspergillus nomius, 7：Sickle
Knife mould Fusarium proliferatum, 8：Water blank control group.
Fig. 7 is the PCR amplifications of 12 plants of geographical strain muscardines based on primer sets F935/R1385.Note：M：
Marker 2000DL；1：Laboratory preserves strain, 2：Flat strain, 3 in Guangxi：The strain of Guangxi Zhong Pinglangdong villages, 4：Period In Maoming strain, 5：
The strain of Guangdong Huazhou, 6：The other strain of Guangxi Yizhou stone, 7：Guangxi Huan Jiang plants, 8：The strain of Guangdong Wengyuan nitre village, 9：Nanning strain, 10：Extensively
Eastern institute of microbiology's biological and ecological methods to prevent plant disease, pests, and erosion strain, 11：Yingde, Guangdong strain, 12：The strain of Hunan Luxi, 13：Water blank group.
Fig. 8 is based on systematic evolution tree gene constructed 12 plants of geographical strain muscardine rps3.
Fig. 9 is PCR amplifications of the primer sets F935/R1385 to different sources stiff silkworm.Note：M：Marker 2000 DL；
No. 1-41 is the commercially available stiff silkworm of different sources；A, No. B be water blank group.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
Limit in any form.
Unless stated otherwise, the present invention is used reagent, method and apparatus for the art conventional reagent, method and are set
It is standby.
Unless stated otherwise, following examples agents useful for same and material are purchased in market.
The muscardine special primer of embodiment 1 is designed
1st, inventor is based on mitochondria full-length genome analytic approach, compares 13 cordyceps sinensis section things including muscardine
The mitochondria whole genome sequence gene structure planted, have found 1 muscardine differential protein encoding gene rps3, and the gene is volume
Code ribosomal protein s3 gene, ribosomal protein S3 is the constitutive protein of ribosomes 40S small subunits, with ribosomes external work
Can, played a significant role during DNA damage reparation, gene expression regulation, Apoptosis etc..
Meanwhile, carry out multisequencing pair using 1.81 softwares of CLUSTAL X, 12 plants of strains system muscardine rps3 genes
Than analysis, as a result find that the moiety site of the rps3 sequences of 12 plants of strains system muscardines has the replacement phenomenon of base
(12 plants of geographical strain muscardine rps3 gene orders comparison results are as shown in Figure 1, 2, 3.Fig. 1,2,3 are continuous successively).
Therefore, Select gene rps3 is applied in Chinese medicine stiff silkworm as the molecular labeling of muscardine mitochondria full-length genome
The muscardine true and false differentiates.
2nd, Guangdong Strain muscardine mitochondria full-length genome is sequenced
Guangdong Strain muscardine chondriogen structure chart is as shown in Figure 4.Guangdong Strain muscardine mitochondria is closed hoop knot
Structure, base total length is 29922bp, and GC ratios are 27.26%, containing 42 encoding genes (15 protein coding genes, 25
TRNA, 2 rRNA), without GAP regions.
Specific method is as follows：
S1. the breeding of Guangdong Strain muscardine strain is with isolating and purifying
PDA culture medium is configured, bred using method of scoring, isolate and purify Guangdong Strain muscardine strain；Prepare Guangdong Strain stiff in vain
Bacterium single bacterium colony.
S2. Guangdong Strain muscardine genome DNA is extracted
Appropriate muscardine single bacterium colony mycelia is taken, liquid nitrogen is added and is fully ground, the use of health is century fungal DNA extraction kits
Sick leaf STb gene is extracted according to its operating instruction.
S3. high-throughput sequencing library is built
S4.Illumina Hiseq2500 platforms are sequenced
S5. quality testing
To ensure the reliability of subsequent analysis, necessary detection and data screening, the content of detection are carried out to sequencing data
Including：Data quality checking, sequencing data amount, sequencing data quality, the distribution of GC ratios, sequencing accuracy etc.；Simultaneously based on inspection
Result is surveyed, data are screened, the standard of screening is：Sequencing data Q20 (ratio of base of the accuracy more than 99%)
It is not less than 83% not less than 90%, Q30 (base ratio of the accuracy more than 99.9%).
S6. mitochondrial genomes sequence capturing
According to issued nearly edge species mitochondrial genomes sequence, the method contrasted based on DNA sequence dna, in STb gene
The mitochondrial genomes sequence of separation sequencing sample in sequencing data.
S7. genome assembling and checking
According to the overlap relations of mitochondrial genomes data, by the DNA sequencing fragment of sequencing, complete line is assembled into
Mitochondrial genes group.And pass through sequencing quality and the correctness of sequencing depth checking assembling.
S8. genome annotation
The annotation of genome is first since tRNA, and using MiTFi, tRNAscan-SE (V1.21) and ARWEN enter to tRNA
Row annotation, it is ensured that the accuracy of tRNA annotations；The annotation of encoding gene and rRNA genes, while based on blastn＆blastx
The method of (2.2.28+) is carried out jointly, is determined by comparing result after specific gene order, then is transcribed into protein sequence
Arrange and annotate to verify whether genome annotation is correct.
S9. evolutionary analysis
Using RAxML (V8.1.5) software maximum likelihood method constructing system chadogram, Bootstrap values are 1000；Using
Optimal nucleosides acid profile be GTR+G+I, optimal nucleosides acid profile be CpREV+I+G+F.
S10. analysis of biological information result collects
Muscardine mitochondrial genomes carry out high-flux sequence using Illumina Hiseq2500 platforms, build PE sequencings
Library, finally collects sequencing data.
Further, according to sequencing and data comparison, constructing system chadogram, as shown in Figure 5, it is determined that Guangdong Strain is stiff in vain
The kind of bacterium is sorted out.
3rd, design of primers
Guangdong Strain muscardine mitochondria rps3 gene high-flux sequence results based on completion, devise multigroup primer, warp
Cross preliminary screening, it is determined that 6 pairs of primer sets further experiments shown in table 1.
Table 1 is based on muscardine mitochondria rps3 genes and designs primer and sequence information
4th, primer is verified
(1) PCR amplification system and program are set up, it is as follows：
The PCR reaction systems of table 2 (50 μ L systems)
PCR reaction conditions：94℃5min；94 DEG C of 30s, 44 DEG C of 30s, 72 DEG C of 45s, 25 circulations；72℃10min.
(2) PCR is expanded
Enter performing PCR amplification with the primer sets shown in table 1, product enters row agarose gel electrophoresis, as a result shown, primer sets I,
That is primer sets F935/R1385, being capable of specific detection muscardine.
Primer sets F935/R1385 PCR amplifications are not as shown in fig. 6, swimming lane 8 (water blank control) occurs reaction bar
Band, illustrates the pollution-free phenomenon of PCR courses of reaction, and reaction result is reliable, and PCR amplifications obtain purpose fragment size in 500bp
The electrophoretic band of left and right.The primer sets F935/R1385 designed based on Guangdong Strain muscardine mitochondria rps3 genes being capable of dialogue deadlock
Bacterium (swimming lane 1,2,3) and muscardine serotype (swimming lane 4) are expanded, and can not to grey stiff bacterium (swimming lane 5), aspergillus (swimming lane 6),
The fungies such as reaping hook mould (swimming lane 7) are expanded, then primer sets F935/R1385 can as quick discriminating muscardine special primer.
The PCR amplifications of the geographical strain muscardine rps3 genes of 2 12 plants of embodiment
1st, using 12 plants of geography strain muscardine DNA as template, enter performing PCR amplification using primer sets F935/R1385, enter one
Step demonstrate,proves the validity of primer.
2nd, result illustrates PCR courses of reaction without dirt as shown in fig. 7, swimming lane 13 (water blank control) does not occur reaction band
Phenomenon is contaminated, reaction result is reliable.The PCR amplifications of 12 plants of geographical strain muscardines have obtained clearly electrophoretic band, mesh
Clip size in 500bp or so.Validation verification to primer is completed by Fig. 7, primer sets F935/R1385 can
Using the special primer as identification muscardine.
3rd, further, it can also continue to amplified production be sequenced and data comparison, constructing system chadogram, further
Determine result.It is as shown in Figure 8 based on 12 plants of gene constructed systematic evolution trees of geographical strain muscardine rps3.
Embodiment 3 differentiates the true and false of muscardine in commercially available stiff silkworm sample based on primer sets F935/R1385
1st, experimental method
(1) commercially available stiff silkworm Genome DNA extraction
Sample pretreatment：Behind 70% ethanol larva of a silkworm with batrytis surface, it is placed in glass dish, and make wine in 37 DEG C of baking ovens
Essence volatilization, is milled with high-speed multifunctional pulverizer, loaded on standby in 25mL sterilizing plastic tubes.
Appropriate stiff silkworm powder is taken, liquid nitrogen is added and is fully ground, using purchased from the true of Shanghai Sheng Gong bioengineering Co., Ltd
Bacterium genome rapid extraction kit extracts muscardine mycelium STb gene, is placed in -20 DEG C and saves backup.
(2) the muscardine true and false differentiates in the commercially available stiff silkworm of different sources
Using Chinese medicine stiff silkworm complete genome DNA as template, performing PCR amplification is entered using primer sets F935/R1385.
2nd, PCR amplifications are tied as shown in figure 9, being expanded by primer sets F935/R1385 to the PCR of different sources stiff silkworm
Fruit is as can be seen that according to the electrophoretic band for whetheing there is clearly 500bp mesh, then may determine that whether contain in commercially available place of production stiff silkworm sample
There is muscardine, so that the true and false to muscardine in the commercially available stiff silkworm of different sources differentiates.
3rd, further, it can also continue to amplified production be sequenced and data comparison, constructing system chadogram, further
Result is determined, the method for identifying molecules differentiated for the muscardine true and false in stiff silkworm provides foundation.
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>The method for identifying molecules of the muscardine true and false in a kind of quick discriminating Chinese medicine stiff silkworm
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>Primers F 935
<400> 1
agtgaatttt ccagatag 18
<210> 2
<211> 18
<212> DNA
<213>Primer R1385
<400> 2
ttccctgata ttcaacct 18
Claims (10)
1. the primer sets of the muscardine true and false in one group of identification checking stiff silkworm, it is characterised in that be primer pair F935 and R1385, sequence
Row are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.
2. application of the primer sets described in claim 1 in identification checking stiff silkworm in the muscardine true and false.
3. primer sets described in claim 1 are in the method for identifying molecules of the muscardine true and false in building quick discriminating Chinese medicine stiff silkworm
Application.
4. the answering in the kit of the muscardine true and false in preparing quick discriminating Chinese medicine stiff silkworm of primer sets described in claim 1
With.
5. the method for identifying molecules of the muscardine true and false in a kind of quick discriminating Chinese medicine stiff silkworm, it is characterised in that with Chinese medicine to be measured
Material larva of a silkworm with batrytis STb gene is template, and performing PCR amplification is entered using primer sets F935/ R1385, and amplified production carries out Ago-Gel electricity
Swimming, the electrophoresis strip that clearly 500bp mesh is whether there is according to PCR amplifications bring judge in Chinese medicine larva of a silkworm with batrytis sample to be measured whether
Contain muscardine.
6. method for identifying molecules according to claim 5, it is characterised in that the extracting method of the larva of a silkworm with batrytis STb gene is：
First the larva of a silkworm with batrytis is pre-processed into after powder, Genome DNA extraction is carried out；The pretreatment refers to：With 65~75% ethanol larva of a silkworm with batrytis
Behind surface, being put in 30~40 DEG C of environment makes alcohol volatilize, then crushes milling.
7. method for identifying molecules according to claim 5, it is characterised in that the reaction system of the PCR is：2xTaq
μ l, the 10pmol/ μ L of Master Mix 25 each 1 μ l of upstream and downstream primer, DNA profiling 1 μ l, ddH2O 2µl；
The reaction condition of the PCR：94℃ 5min；94 DEG C of 30s, 44 DEG C of 30s, 72 DEG C of 45s, 25 circulations；72℃
10min。
8. method for identifying molecules according to claim 5, it is characterised in that can also continue to pcr amplification product
It is sequenced and data comparison, constructing system chadogram further determines that result.
9. the kit of the muscardine true and false in a kind of quick discriminating Chinese medicine stiff silkworm, it is characterised in that include claim 1 institute
The primer pair F935 and R1385 stated.
10. kit according to claim 9, it is characterised in that also include using Chinese medicine larva of a silkworm with batrytis STb gene to be measured as
Template, the reagent entered using primer sets F935/ R1385 needed for performing PCR amplification.
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Cited By (4)
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| CN107937567A (en) * | 2017-12-29 | 2018-04-20 | 江苏大学 | The specific primer and its identification method of one group of identification stiff silkworm |
| CN112730291A (en) * | 2020-12-23 | 2021-04-30 | 江苏省中医院 | Qualitative and quantitative marker of silkworm counterfeit product and detection method thereof |
| CN116068096A (en) * | 2023-04-04 | 2023-05-05 | 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 |
| WO2023082512A1 (en) * | 2021-11-10 | 2023-05-19 | 广东一方制药有限公司 | Stiff silkworm marker polypeptide and method for identifying stiff silkworms, stiff silkworm aqueous extract products and other stiff silkworm products |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937567A (en) * | 2017-12-29 | 2018-04-20 | 江苏大学 | The specific primer and its identification method of one group of identification stiff silkworm |
| CN107937567B (en) * | 2017-12-29 | 2020-09-25 | 江苏大学 | Specific primers for identifying bombyx batryticatus and identification method thereof |
| CN112730291A (en) * | 2020-12-23 | 2021-04-30 | 江苏省中医院 | Qualitative and quantitative marker of silkworm counterfeit product and detection 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 |
| WO2023082512A1 (en) * | 2021-11-10 | 2023-05-19 | 广东一方制药有限公司 | Stiff silkworm marker polypeptide and method for identifying stiff silkworms, stiff silkworm aqueous extract products and other stiff silkworm products |
| CN116068096A (en) * | 2023-04-04 | 2023-05-05 | 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 |
| 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 |
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| CN107164471B (en) | 2020-08-07 |
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