A molecular identification method for Isaria cicadae

A technology for molecular identification of Cicada cicada, applied in the field of molecular biology, can solve problems such as multiple identification errors

Inactive Publication Date: 2013-01-02
ZHEJIANG BIOASIA PHARMA CO LTD
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Problems solved by technology

[0007] Due to the different sources of geographical environment and different hosts, the secondary metabolites of I. cicadae strains are quite different, and this difference is difficult to distinguish by traditional identification methods, because the traditional methods are difficult for the identification of entomogenic fungi and intraspecific The analysis of variation and strain c

Method used

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  • A molecular identification method for Isaria cicadae
  • A molecular identification method for Isaria cicadae
  • A molecular identification method for Isaria cicadae

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Experimental program
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Embodiment 1

[0040] experiment procedure:

[0041] 1) After cultivating I. cicadae CGMCC NO.3453 strain by conventional methods, extract the genomic DNA of the strain;

[0042] Genomic DNA extraction method:

[0043] Using the conventional method of chloroform extraction and isopropanol precipitation of DNA, the liquid cultured mycelium was washed twice with sterilized water, and then suspended in TEN-3 containing 3M guanidine isothiocyanate (50mM Tris-Hcl, 100mM EDTA, 50mM Nacl, PH=8.3) solution, after the homogenate was broken on ice bath, add 700ul extract solution (50mM Kcl, 10mM Tris-Hcl, PH=8.3, 2.5mMMgcl 2 , 0.1mg / ml gelatin, 0.45% NP40, 0.45% Tween 20) add 6ul (10mg / m1) proteinase K, mix well and place in a water bath at 55°C for 1 hour, and extract twice with an equal amount of chloroform, Add an equal amount of isopropanol, place in -20°C refrigerator overnight, then centrifuge at 15,000 r / min for 15 minutes (4°C) to collect the precipitate, wash once with 70% alcohol, dissolve...

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Abstract

The invention relates to the field of biotechnology and discloses a molecular identification method for Isaria cicadae (CGMCCNO.3453). The method includes the steps of extracting genomic DNA of the strain, performing amplification of the DNA in the transcription spacers of the genome using primers ITS5 and ITS4, and recovering and purifying the obtained DNA fragments; performing bidirectional sequencing of the purified DNA fragments; aligning the obtained DNA sequences with SEQIDNO:3; and identifying the strain as Isaria cicadae (CGMCCNO.3453) if the ITS sequence of the strain has the same content and order of arrangement as SEQIDNO:3. The inventive method can rapidly, reliably and accurately identify Isaria cicadae (CGMCCNO.3453), and the identification method is not influenced by environmental factors.

Description

technical field [0001] The invention relates to the technical field of molecular biology, and in particular relates to the establishment of a molecular biology identification method for a strain of I. cicadae (preservation number: CGMCC NO.3453). Background technique [0002] Cicada is the common name of Isaria cicadae Miquel (Isaria cicadae Miquel=Paecilomyces cicadae (Miq.) Samson), which is formed after some cicada nymphs are parasitized by Cicada The product is a complex of bacteria and insects, named by Miquel in 1838. [0003] The Isaria cicadae Miquel strain (Isaria cicadae Miquel) involved in this patent has been registered and preserved in the General Microorganism Center (CGMCC) of China Microbiological Culture Collection Management Committee (abbreviated as CGMCC) on November 18, 2009, and the preservation number is CGMCC NO.3453. [0004] This bacterial strain is collected wild cicadae specimens from the source of Sanmen, Yunnan, and is artificially cultured and...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 鲍晓妮陈超魏宁张健江三多孙长胜
Owner ZHEJIANG BIOASIA PHARMA CO LTD
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