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Specific primer for identifying poecilobdella manillensis based on molecular biological method

A molecular biology and specific technology, applied in the field of molecular biology, can solve the problems of difficult and time-consuming identification of Hirudo phenanthae medicinal materials, and achieve the effect of reducing the time of comparative analysis, reducing operation time, and reducing professional restrictions

Inactive Publication Date: 2018-11-23
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, the identification methods of Hirudo phenifundii mainly rely on the identification of traits and molecular biology. It is quite difficult. The current molecular biology identification method is the method of DNA barcode. It is necessary to sequence the amplified products of the universal primers and compare and analyze the sequenced sequences, which takes a long time and requires operators to be familiar with molecular biology. Knowledge and familiarity with the use of professional software

Method used

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  • Specific primer for identifying poecilobdella manillensis based on molecular biological method
  • Specific primer for identifying poecilobdella manillensis based on molecular biological method
  • Specific primer for identifying poecilobdella manillensis based on molecular biological method

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Effect test

Embodiment 1

[0027] A pair of specific primers based on specific amplification of molecular biology to identify Hirudo phenanthrae, the upstream primer is: 5'-TCTGGCTCTTAGGCTTCG-3', the downstream primer is: 5'-GTTGTCTTCCTGGCTGTT-3', provided by Shanghai Jierui Bioengineering Co., Ltd. Company Synthesis.

[0028] Using the above primers to quickly identify Hirudo phenanthae, the samples to be tested were selected from the leech samples numbered SZ-1~SZ-20 in Table 1, and parallel experiments were carried out to identify whether the leech samples numbered SZ-1~SZ-20 were Hirudo phenatus. ;Specific steps are as follows:

[0029] (1) Extraction of total DNA: Take 20-30 mg of the sample to be tested, grind the dry product with MM400 ball mill, cut the fresh sample with surgical scissors, and use the DNeasy Blood & Tissue Kit genome extraction kit from QIAGEN Company to perform DNA extraction;

[0030] (2) Polymerase chain reaction: 20 μL of PCR reaction system includes 10 μL of 2×Taq Master M...

Embodiment 2

[0035] The annealing temperature of the specific primers in Example 1 was investigated. A total of 6 annealing temperatures of 45°C, 47°C, 49°C, 51°C, 53°C, and 55°C were investigated. According to the investigation results, 49°C was determined to be The optimal annealing temperature of primer 7 to specifically amplify the sequence of B. phenanthrene. Specific steps are as follows:

[0036] (1) Extraction of total DNA: randomly select three batches of samples numbered SZ-10, SZ-14, and SZ-18, take 20-30 mg of the sample to be tested, grind the dry product with MM400 ball mill, and cut the fresh sample with surgical scissors , using QIAGEN's DNeasy Blood&Tissue Kit genome extraction kit for DNA extraction;

[0037] (2) Polymerase chain reaction: 20 μL of PCR reaction system includes 10 μL of 2×Taq Master Mix buffer, 0.4 μL of 25 μmol / L upstream and downstream primers, 1 μL of 50 ng / μL DNA template, and 8.2 μL of sterilized double-distilled water. The PCR reaction conditions a...

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Abstract

The invention discloses a specific primer for identifying poecilobdella manillensis based on a molecular biological method. An upstream primer is 5'-TCTGGCTCTTAGGCTTCG-3', and a downstream primer is 5'-GTTGTCTTCCTGGCTGTT-3'. A method for quickly identifying poecilobdella manillensis based on amplification of the specific primer comprises the following main steps: (1) extracting total DNA of a sample to be detected to obtain extracted genome DNA; (2) performing PCR (Polymerase Chain Reaction) amplification by taking the genome DNA extracted in the step (1) as a template and adopting the upstream primer and the downstream primer according to claim 1; and (3) performing agarose gel electrophoresis detection and gel imaging on the reaction product obtained in the step (2) to realize specific identification of poecilobdella manillensis, thereby providing a reference for a molecular biological identification method of poecilobdella manillensis.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a specific primer for identifying Hirudo phenanthae based on molecular biology methods. Background technique [0002] The Philippine leech (Poecilobdella manillensis) is commonly known as the Phnom Penh leech, also known as the Mani pseudo-medical leech. Its chemical components mainly include a variety of fatty acids, amino acids and trace elements. The active ingredients are mainly hirudin, phenanthrene A, phenanthrene B. In addition, Hirudo phenanthae also contains some other biologically active ingredients, such as anticoagulant polypeptides, fibrinolytic enzymes, hyaluronidase, vasodilators, anti-platelet aggregation active ingredients, antibacterial active ingredients, etc. The pharmacological effects of Hirudo phenanthae mainly include anti-platelet aggregation and anticoagulant effect, inhibition of thrombosis, lipid-lowering effect, anti-tumor effect...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2565/125
Inventor 郑健刘杰过立农昝珂马双成
Owner NAT INST FOR FOOD & DRUG CONTROL
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