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Specific primers and method for identifying stiff silkworms

A specific and silkworm technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of inability to detect adulterated products, many steps, and time-consuming, etc., to achieve exclusive Strong, simple operation, no sequencing effect

Active Publication Date: 2018-04-20
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of species identification using DNA sequence by ordinary PCR technology, due to the use of universal primers, after the amplification is completed, the product needs to be sequenced and compared with the gene database to determine the species, which requires many steps and consumes a lot of time. It takes a long time and requires special equipment; in addition, it is impossible to detect adulterated products, or the identification results are affected by adulteration

Method used

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  • Specific primers and method for identifying stiff silkworms
  • Specific primers and method for identifying stiff silkworms
  • Specific primers and method for identifying stiff silkworms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Design and screening of specific primers

[0047] (1) Reagent

[0048] Tris balanced phenol (Solarbio LIFE SCIENCES T0250), disodium edetate, chloroform, isoamyl alcohol, absolute ethanol, sodium lauryl sulfate, sodium chloride, trishydroxymethylaminomethane, boric acid, Dream Taq Green DNA Polymerase (Thermo Scientific #EP0712), dNTP Mix (Thermo Scientific #R0192), Agarose (SunShineBio A0009-3), Ethidium Bromide (SunShineBio SN314-1). All reagents were of analytical grade, and primers were synthesized by Shanghai Sangon Co., Ltd.

[0049] (2) method

[0050] a. Design specific primers:

[0051] Search and download silkworm in GenBank database (GU966628.1, GU966626.1, GU966625.1, GU966624.1, GU966623.1, GU966622.1, GU966620.1, GU966618.1, GU966617.1, GU966615.1, GU96 1. GU966613.1, GU966612.1, GU966611.1, GU966610.1, GU966609.1, GU966608.1, GU966607.1, GU966605.1, GU966604.1, GU966603.1, GU966602.1, GU966602.1, GU966602.1, GU966603.1 GU966599.1, GU966596...

Embodiment 2

[0058] Example 2: Application of Bombyx mori kit primers in self-made Bombyx mori samples

[0059] With reference to the 15th edition of the Pharmacopoeia, 10 batches of dry bodies infected (or artificially inoculated) with Beauveria bassiana by 4-5 instar larvae of Bombyx mori, Bombyx mori, were pulverized and used as spare silkworm medicinal materials. 10 batches of dry bodies infected (or artificially inoculated) with Metarhizium anisopliae by 4-5 instar larvae of Bombyx mori, Bombyx mori, were pulverized and used as counterfeit Bombyx mori medicinal materials.

[0060] Different batches of self-made silkworm samples were pulverized, 10 mg of the sample powder was weighed, extracted and purified by the CTAB method to prepare the test solution, and the following steps were carried out:

[0061] (1) Accurately weigh 10 mg to 60 mg of silkworm powder, add 500 μL to 1000 μL of 2×CTAB extract, bathe in water at 55°C to 65°C for 1h to 6h, centrifuge at 12000r / min for 10min, and t...

Embodiment 3

[0073] Embodiment 3: The specific primer of the present invention is to the detection of actual commercially available silkworm sample

[0074] Different batches of commercially available silkworm decoction pieces were pulverized, and 10 mg of sample powder was weighed, and extracted and purified by CTAB method according to the method of Example 2. Thereafter, prepare a PCR reaction system, place it in a PCR instrument, and perform amplification according to the above-mentioned optimized conditions. Subsequently, 8 μL of the amplified product was analyzed using 2.0% agarose gel containing EB and TBE buffer. After electrophoresis, it was detected by a gel imaging system, and the presence or absence of electrophoresis bands and the molecular weight were used to determine the authenticity. In addition, the positive control substances of silkworm, Beauveria bassiana and Metarhizium anisopliae weighed 10 mg of sample powder respectively, extracted and purified by CTAB method and q...

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Abstract

The invention relates to a group of specific primers and a method for identifying stiff silkworms, belonging to the technical field of identification of traditional Chinese medicines. According to themethod, after a stiff silkworm sample is crushed, the deoxyribonucleic acid (DNA) in domestic silkworms and the DNA in fungi are extracted at the same time by using a CTAB method; the extracted DNA is taken as a template, specific nucleotide sequences, which are designed and screened, are used as primers for performing polymerase chain reaction (PCR) amplification, and the products are then subjected to agarose gel electrophoresis analysis and gel imaging system detection, so that the authenticity and adulteration of the stiff silkworms can be finally detected based on the detection results.The method provided by the invention can be used for judging whether the sample is a stiff silkworm fake product or adulterated product, is simple to operate, does not needing sequencing, is high in specificity, rapid and accurate, and has unique advantages and important application value in the authenticity identification of stiff silkworm samples.

Description

technical field [0001] The invention relates to a group of specific primers for identifying Bombyx mori and an identification method thereof, belonging to the field of identification of traditional Chinese medicines. Background technique [0002] Bombyx mori is a dry body that is lethally infected (or artificially inoculated) with Beauveria bassiana (Bals.) Vuilknt by the 4th to 5th instar larvae of Bombyx mori Linnaeus. It is more than spring and autumn production, and the silkworms infected with Beauveria bassiana are dried. Silkworm was first recorded in "Shen Nong's Materia Medica", and has a long history of application. It is salty, pungent, flat, and belongs to the liver, lung, and stomach meridian. Modern pharmacological studies have shown that Bombyx mori has anticoagulant, anticonvulsant, anticancer, sedative and hypnotic effects. [0003] Bombyx mori is a common traditional Chinese medicine for animals. It has reliable curative effect and high medicinal value. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6895C12Q1/686C12N15/10C12N15/11
CPCC12N15/1003C12Q1/686C12Q1/6888C12Q1/6895C12Q2565/125
Inventor 杨欢周莹沈玉萍杨娅娅吴啟南夏国华陈宇菲鹿蓓蓓
Owner JIANGSU UNIV
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