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Method and primer for fast detecting Cronobacter sakazakii at constant temperature and application

A technology of Cronobacter sakazakii, applied in the field of rapid constant temperature detection of Cronobacter sakazakii, which can solve the problems of lack of generality and specificity of primers

Active Publication Date: 2017-02-22
DRAGONS MORAL SHANGHAI ADMINISTATION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the defects of primer versatility and specificity in the primer design of the existing LAMP technology, make full use of the abundant microbial genome sequence information and corresponding sequence analysis tools in the current public data resources, and design A primer set for specific recognition of Cronobacter sakazakii, and on this basis, a high-sensitivity and high-specificity detection kit

Method used

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  • Method and primer for fast detecting Cronobacter sakazakii at constant temperature and application
  • Method and primer for fast detecting Cronobacter sakazakii at constant temperature and application
  • Method and primer for fast detecting Cronobacter sakazakii at constant temperature and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1-6

[0187] Embodiment 1-6 Cronobacter sakazakii constant temperature reaction system and detection method

[0188] Follow the steps (1) to (3) below for testing:

[0189] (1) Extraction of genomic DNA

[0190] The strains of Cronobacter sakazakii used for the detection were obtained from the China Center for the Collection of Industrial Microorganisms, numbered CICC21560 (=ATCC29544). Take 1mL of bacterial culture and use Beijing Tiangen Bioengineering Company's Bacterial Nucleic Acid Extraction Kit to extract genomic DNA, DNA OD 260 / OD 280 It was 1.8, and the concentration was 288ng / μL.

[0191] (2) Using the genomic DNA of Cronobacter sakazakii to be tested as a template, using self-prepared kits (see Table 2, Table 3) respectively, and preparing a reaction system according to the conditions described in Table 3, using Gram Sakazaki The Rhinobacter specific amplification primer set is used as primers for constant temperature amplification reaction. The primers in Examples ...

Embodiment 7

[0195] Example 7 Specific detection of Cronobacter sakazakii

[0196] Collect 28 strains of non-Sakazakii Cronobacter (Table 4 and figure 1 1~22, 24~29 in ), these strains were compared with Cronobacter sakazakii strains (Table 4 and figure 1 23) in 23) are cultured respectively, get 1mL bacterium liquid, adopt kit 1A, extract bacterial DNA, and with reference to the reaction system and condition of embodiment 1, carry out LAMP amplification respectively (primer group is A) and add chromogenic agent to observe .

[0197] The test results are shown in Table 4 and figure 1 as shown, figure 1 Among them, 1-22 are Staphylococcus aureus, Staphylococcus aureus subsp. aureus, Staphylococcus epidermidis, Rhodococcus equi, Bacillus cereus, Bacillus mycoides, Listeria monocytogenes, Listeria innoculus Tertiary bacteria, Listeria evanii, Salmonella enterica subsp. enterica, Salmonella enteritidis, Salmonella typhimurium, Salmonella paratyphi B, Shigella dysenteriae, Shigella baumanni...

Embodiment 8

[0201] Embodiment 8 Sensitivity detection

[0202] Extract the DNA of bacteria CICC 21560 according to the method of Example 2, adopt kit IIB, and add reaction system according to 50ng, 5ng, 500pg, 50pg, 5pg, 500fg, 50fg DNA gradient, other reaction conditions refer to the method of Table 3 Example 2 Carry out LAMP amplification (primer set is B') and add chromogen for observation respectively. Such as figure 2 As shown, 1-7 are 50ng, 5ng, 500pg, 50pg, 5pg, 500fg and 50fg respectively, NTC: negative control. figure 2 The reaction products treated with 50ng, 5ng, 500pg, 50pg, and 5pg were bright green, which was a positive result, and the reaction products treated with 500fg and 50fg and the negative control were orange, which was a negative result. The test results show that each reaction tube can still be detected when the minimum DNA content is 5pg (equivalent to about 1000 bacteria), and the sensitivity is high.

[0203] According to the above detection method, other s...

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Abstract

The invention discloses a method, primer set and kit for fast detecting Cronobacter sakazakii at constant temperature. The method includes the steps that genome DNA is extracted from a sample to be detected; with the genome DNA as a template, the primer set capable of amplifying the specific sequence of Cronobacter sakazakii is used as the primer, and constant-temperature amplification reaction is carried out under the enzyme reaction system; whether the reaction result is positive or not is judged to determine whether Cronobacter sakazakii exists in the sample to be detected. The detection method is high in sensitivity and specificity, short in detection time, convenient to operate and low in cost and has wide application prospects, and the result is easy to judge.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a method, primers and kit for rapid and constant temperature detection of Cronobacter sakazakii. Background technique [0002] Cronobacter sakazakii (Cronobacter sakazakii) is a Gram-negative, peri-flagellated, motile, non-spore-forming facultative anaerobic bacterium, known as atypical yellow pigment-producing Enterobacter cloacae before 1980, After 1980, it was renamed as Enterobacter sakazakii, and it was reclassified and named in 2008, and it was assigned to the genus Cronobacter in the family Enterobacteriaceae. Cronobacter sakazakii widely exists in nature and is an important opportunistic pathogen that endangers the health of infants and young children through formula powder. It can cause serious clinical symptoms in infants, such as brain abscess, meningitis, necrotizing enterocolitis and systemic Sexual sepsis, etc., the fatality rate is as high as 40%-8...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2531/119Y02A50/30
Inventor 曹永梅李雪玲刘伟李园园韦朝春陆长德李亦学贾犇吕晓冬陈霞
Owner DRAGONS MORAL SHANGHAI ADMINISTATION
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