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A specific pcr method for rapid identification of intermediate thermoactinomycetes

A high-temperature actinomycetes, fast technology, applied in the field of molecular biology identification, can solve the problems of cumbersome and time-consuming identification of conventional technology, unable to meet the requirements of intermediate type high-temperature actinomycetes screening and application research, etc., to achieve convenient cost, primers The effect of strong specificity and simple PCR amplification conditions

Active Publication Date: 2015-08-05
CHINA NAT RES INST OF FOOD & FERMENTATION IND CO LTD +1
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  • Abstract
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  • Application Information

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Problems solved by technology

However, the morphological characteristics, physiological and biochemical characteristics, and 16S rRNA gene sequence of intermedium thermoactinomycetes and their relatives are very similar, and the identification by conventional techniques is cumbersome and time-consuming, which cannot meet the requirements of screening and intermediary hyperthermia actinomycetes. Therefore, there is an urgent need to establish a set of accurate and rapid identification methods for intermediate high-temperature actinomycetes, so as to provide technical support for the breeding of strains and their application research.

Method used

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  • A specific pcr method for rapid identification of intermediate thermoactinomycetes
  • A specific pcr method for rapid identification of intermediate thermoactinomycetes

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Embodiment 1

[0024] Embodiment one: the design of specific PCR primer

[0025] According to the intermediate-type high-temperature actinomycetes publicly registered on GenBank gyrB Gene sequence information for primers

[0026] design (see attached figure 1 ).

[0027] The GenBank accession number of the reference sequence is: HM595857 ( Thermoactinomyces intermedius , gyrB gene, http: / / www.ncbi.nlm.nih.gov / nuccore / 82568126 ）。

[0028] An upstream primer was designed at 61-78 and named 78F. The sequence is: 5'-TTGTCCACGTGGTTGTCG-3'.

[0029] A downstream primer was designed at 618-637 and named 618R. The sequence is: 5'-TACGTCATCCCCGGTCAG-3'.

Embodiment 2

[0030] Example 2: Primer Specificity Verification

[0031] 1.1 Strains of intermediate-type hyperthermic actinomycetes and their relative strains

[0032] common high temperature actinomycetes ( Thermoactinomyces vulgaris DSM 43016), Thermoactinomycetes intermediate ( Thermoactinomyces intermedius DSM 43816), Laysia saccharum ( Laceyella sacchari DSM 43356), Laceyella putida ( Laceyella putidus DSM44608) was purchased from the German Culture Collection of Microorganisms; Bacillus thuringiensis ( Bacillus thuringiensis CICC 22945), Bacillus licheniformis ( Bacillus licheniformis CICC 10101) from China Industrial Microorganism Culture Collection Management Center; Laceyella tengchongensis CCTCC AA 208050, Laceyella sediminis CCTCC AA 2011024 was purchased from China Type Culture Collection Center; Thermoactinomyces vulgaris ACCC 41061) was purchased from the China Agricultural Microorganism Culture Collection Management Center; Thermoactinomyces vulgaris ...

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Abstract

The invention discloses a specific PCR method for quickly identifying intermediate-type high-temperature actinomycetes. The technical scheme uses genomic DNA as a template, uses the specific primers designed by the invention to carry out PCR reaction, and amplifies the characteristics of intermediate-type high-temperature actinomycetes The housekeeping gene, the actual amplified fragment is 576bp, and the amplified product is detected by agarose gel electrophoresis to identify whether the sample is an intermediate type of high-temperature actinomycetes. The invention can quickly identify the intermediate high-temperature actinomycetes, and only needs three steps of DNA extraction, PCR amplification and electrophoresis detection to complete the identification, and has significant advantages such as high efficiency, sensitivity, convenience and low cost.

Description

Technical field [0001] The present invention is the field of molecular biology identification, and it involves a fast identification method of intermediate high -temperature release bacteria. Background technique [0002] Intermediate high -temperature release bacteria ( Thermoactinomycess intermedius ) The earliest was discovered by Kurup in 1981. It belongs to the bacterium-Firmicutes-Bacilli-Bacillales-ThermoActinomyCetaceae-High temperature releaseThe genus (Thermoactinomycess), which is common in the high temperature environment of nature, is addicted to high temperature, which can produce richcers and endogenous spores. [0003] The intermediate high -temperature release bacteria can produce heat -resistant, light -stable glitterine dehydrogenase and phenylalanine dehydrogenase, which has important application value in the drug industry.In recent years, sesame -fragrant liquor brewing microorganisms have found that intermediate high -temperature release bacteria are the adv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/04C12Q1/686C12Q2531/113
Inventor 程池赵纪文姚粟信春晖李辉刘勇张明娟
Owner CHINA NAT RES INST OF FOOD & FERMENTATION IND CO LTD
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