Molecular identifying method for quickly identifying truth of beauveria bassiana in Chinese herbal medicine stiff silkworm

A technology for molecular identification and Beauveria bassiana is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial determination/testing, which can solve the problem of lack of molecular identification methods for rapid identification of Beauveria bassiana and the lack of specific verification. Questions about the primers of B.

Active Publication Date: 2017-09-15
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, in the identification of the authenticity of Beauveria bassiana in silkworm, there has been a lack of primers for specific verification

Method used

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  • Molecular identifying method for quickly identifying truth of beauveria bassiana in Chinese herbal medicine stiff silkworm
  • Molecular identifying method for quickly identifying truth of beauveria bassiana in Chinese herbal medicine stiff silkworm
  • Molecular identifying method for quickly identifying truth of beauveria bassiana in Chinese herbal medicine stiff silkworm

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 Beauveria bassiana specific primer design

[0042] 1. Based on the mitochondrial whole genome analysis method, the inventor compared the mitochondrial whole genome sequence gene structure of 13 Cordyceps species including Beauveria bassiana, and found a differential protein coding gene rps3 of Beauveria bassiana, which is The gene encoding ribosomal protein s3, ribosomal protein S3 is a constituent protein of the ribosomal 40S small subunit, has extra-ribosome functions, and plays an important role in DNA damage repair, gene expression regulation, and cell apoptosis.

[0043] At the same time, using CLUSTAL X 1.81 software, 12 different geographical strains of Beauveria bassiana rps3 genes were used for multiple sequence comparison analysis, and the results found that there were base substitutions in some sites of the rps3 sequences of 12 different geographical strains of Beauveria bassiana (12 The sequence alignment results of Beauveria bassiana rps3 gene ...

Embodiment 2

[0079] Example 2 PCR amplification of 12 geographical strains Beauveria bassiana rps3 gene

[0080] 1. Using the DNA of 12 geographical strains of Beauveria bassiana as templates, the primer set F935 / R1385 was used for PCR amplification to further verify the effectiveness of the primers.

[0081] 2. The result is as follows Figure 7 As shown, there is no reaction band in swimming lane 13 (water blank control), indicating that there is no pollution in the PCR reaction process, and the reaction result is reliable. The PCR amplification results of 12 geographic strains of Beauveria bassiana all obtained clear electrophoresis bands, and the target fragments were all about 500bp in size. pass Figure 7 The validity verification of the primers has been completed, and the primer set F935 / R1385 can be used as specific primers for identifying Beauveria bassiana.

[0082] 3. Further, the amplified products can also be sequenced and data compared, and a phylogenetic tree can be const...

Embodiment 3

[0083] Example 3 Based on the primer set F935 / R1385 to identify the authenticity of Beauveria bassiana in commercially available Bombyx mori samples

[0084] 1. Experimental method

[0085] (1) Total DNA extraction from commercially available Bombyx mori

[0086] Sample pretreatment: wipe the surface of the silkworm with 70% ethanol, put it in a glass dish, and volatilize the alcohol in an oven at 37°C, grind it into powder with a high-speed multifunctional pulverizer, and put it in a 25mL sterilized plastic tube for later use.

[0087] Take an appropriate amount of silkworm powder, add liquid nitrogen to fully grind, use the fungal genome rapid extraction kit purchased from Shanghai Sangong Bioengineering Co., Ltd. to extract the total DNA of Beauveria bassiana mycelium, and store it at -20°C for future use.

[0088] (2) Identification of the authenticity of Beauveria bassiana in silkworms sold in different places of origin

[0089] Using the whole genome DNA of Bombyx mori...

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Abstract

The invention discloses a primer set for quickly identifying truth of beauveria bassiana in Chinese herbal medicine stiff silkworm and a molecular identifying method. The sequence of the primer set is respectively shown as SEQ ID NO.1-2. According to the invention, on the basis of the completion of high-throughput sequencing of complete genome of Guangdong beauveria bassiana mitochondria and bioinformatic comparative analysis of mitochondria complete genome of multiple cordyceps species, the difference of the beauveria bassiana mitochondria gene sequences at the aspect of geographical species is compared and obvious variation of ribosomal protein gene of beauveria bassiana is founded; a pair of primers for specifically detecting beauveria bassiana are screened on the basis of the design; the molecular identifying method for quickly and efficiently identifying truth of beauveria bassiana in Chinese herbal medicine stiff silkworm is established; the method is simple, convenient and quick, has high specificity, high sensitivity and wide application scope and can realize the identification for the truth of the beauveria bassiana from different producing places; a technical support is supplied for quality standardization and normalization and clinic drug safety of the Chinese herbal medicine stiff silkworm.

Description

technical field [0001] The invention belongs to the technical field of species identification and authenticity identification of Chinese medicinal materials. More specifically, it relates to a molecular identification method for rapidly identifying the authenticity of Beauveria bassiana in silkworm Bombyx mori. Background technique [0002] Bombyx mori, Bombyx mori Linnaeus 4-5 instar larvae infected (or artificially inoculated) Beauveria bassiana (Bals.) Vuillant dead body (National Pharmacopoeia Commission, 2015 ). Beauveria bassiana in B. bassiana is a type of pathogenic fungus that parasitizes insects. According to the latest data from NCBI, Beauveria bassiana is classified into: Dikarya, Ascomycota, and Panmycota. (Pezizomycotina), Sordariomycetes, Hypocreonycetidae, Hypocreales, Cordycipitaceae, Cordceps. [0003] As a traditional Chinese medicinal material in my country, white silkworm has various pharmacological effects such as hypoglycemic, hypolipidemic, antican...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q2600/156
Inventor 刘吉平刘健霞陈杰湖李香霖何青李峙贤刘希吕思行米红霞
Owner SOUTH CHINA AGRI UNIV
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