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Cronobacter spp. standard strain containing specific molecular target and detection and application of cronobacter spp. standard strain

A Cronobacter, molecular target technology, applied in the detection of Cronobacter's molecular target and its standard bacteria field, can solve the problem of high misjudgment rate, can not well reflect the transmission characteristics of the bacteria, Cronobacter detection. Problems such as low rate

Active Publication Date: 2021-06-04
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, most of the standard strains used to study the evolution and pathogenicity of this bacteria are strains from foreign clinical sources, which cannot well reflect the characteristics of the spread of this bacteria in food, and China is an important food production and consumption country in the world. , the detection rate of Cronobacter has been at a high level, and the misjudgment rate of this bacterium is high, and there is still a lack of representative standard strains to study the genetic structure and spread of this bacterium in China

Method used

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  • Cronobacter spp. standard strain containing specific molecular target and detection and application of cronobacter spp. standard strain
  • Cronobacter spp. standard strain containing specific molecular target and detection and application of cronobacter spp. standard strain
  • Cronobacter spp. standard strain containing specific molecular target and detection and application of cronobacter spp. standard strain

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Embodiment 1

[0035] The isolation and cultivation of embodiment 1 Cronobacter standard bacterial strain

[0036] The collected food samples were shredded thoroughly under aseptic conditions, and 25 g of each sample was weighed and added to 225 mL of modified lauryl sulfate tryptose broth-vancomycin (modified lauryl sulfate tryptose broth-vancomycin edium, mLST- Vm) in a homogenizing bag, homogenize continuously for 1-2 minutes on a slap-type homogenizer, and make a uniform dilution of 1:10. Use a pipette to draw 1mL of the 1:10 dilution, add it into a test tube containing 9mL mLST-Vm, shake it well, and prepare a 1:100 dilution. In addition, prepare 10 incremental dilutions at a time according to the above operation, and use a 1mL sterilized pipette tip for each increment, inoculate three test tubes containing 9mL mLST-Vm for each dilution, inoculate 1mL in each tube, and place at 37°C In the incubator, cultivate for 8h-18h. In addition, the remaining mixture was placed in an incubator...

Embodiment 2

[0041] Physiological and biochemical characteristics and serotype analysis of the Cronobacter standard strain of embodiment 2

[0042] Staining microscopy: smear suspicious colonies, perform Gram staining, and observe under an oil lens or a phase-contrast microscope. Cronobacter is a Gram-negative non-spore-forming bacterium with perinatal flagella about 3 μm in length and 1 μm in diameter. (figure 1)

[0043] Biochemical identification: pick a single suspicious colony of pure culture, carry out oxidase test, the bacterium colony of oxidase negative reaction is identified by AIP20E identification system ( figure 2 ).

[0044] Molecular identification: Cronobacter carries the fusA gene and is biochemically identified as Enterobacter sakazakii. The fusA gene amplification test was carried out with the following primers. For the method, see (https: / / pubmlst.org / cronobacter / ). The amplified products were sequenced, and the species were determined by blast comparison analysis...

Embodiment 3

[0046] The drug susceptibility characteristic of embodiment 3 Cronobacter standard strains

[0047] Drug susceptibility test: After the strain was activated on a TSA plate, it was diluted with normal saline to a final concentration of 1×10 7 Cfu / mL was spread on the MH plate, and after the bacterial solution was dry, the antibiotic paper was pasted on the surface of the culture medium, and incubated at 37°C for 24 hours. The size of the inhibition zone was measured with a vernier caliper, accurate to 0.01mm. The selected antibiotics were as follows: ampicillin (AMP, 10 μg), cefazolin (KZ, 30 μg), cephalothin (KF, 30 μg), gentamicin (CN, 10 μg), tobramycin (TOB10), amida Carcin (AK30), ampicillin-sulbactam (SAM10), cefepime (FEP30), ceftriaxone (CRO30), ciprofloxacin (CIP, 5μg), imipenem (IPM10), trimethoprim Pyridine-sulfamethoxazole (SXT, 25 μg), aztreonam (ATM30), chloramphenicol (C, 30 μg), tetracycline (TE30), amoxicillin-clavulanic acid (AMC30). Escherichia coli ATC...

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Abstract

The invention relates to the technical field of bioengineering, in particular to four cronobacter spp. standard strains which have proprietary intellectual property rights in China and conform to the domestic propagation law, and the preservation numbers of the four cronobacter spp. standard strains are respectively GDMCC 60861, GDMCC 60862, GDMCC 60863 and GDMCC 60864. The four standard strains have typical physiological and biochemical characteristics, are clear in information such as sample sources, genetic backgrounds, drug resistance and the like, and can be used as standard reference strains in different fields such as foods, medicines and clinical examination. The invention also relates to a group of specific target genes for detecting and identifying the four standard strains and corresponding PCR products. Finally, the invention also provides a cronobacter spp. freeze-drying protective agent with high survival rate and strong specificity, and the cronobacter spp. freeze-drying protective agent can be used for long-term storage of the standard strains.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to the detection of molecular targets of Cronobacter and standard bacteria thereof. Background technique [0002] Cronobacter spp. (Enterobacter sakazakii) is a facultative anaerobic Gram-negative non-bacillus, which is an important food-borne pathogenic bacterium that can infect people of all ages , especially the elderly and newborn infants with weakened immunity. Infecting infants and young children can cause serious diseases such as necrotizing enterocolitis, bacteremia, and meningitis, with a fatality rate as high as 40% to 80%. There are 7 species of this genus and they are widely present in the environment, and the contamination rate in various foods is also high, especially in vegetables and cooked food. In recent years, there are often reports of Cronobacter being detected in food for infants and young children, which has caused serious losses to dairy ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12N1/20C12N1/04C12R1/01
CPCC12Q1/689C12N1/04Y02A50/30
Inventor 曾海燕吴清平张菊梅丁郁陈谋通薛亮王涓叶青华吴诗陈惠元雷涛古其会杨小鹃张淑红卢勉飞蔡芷荷徐环李程思陈鲁
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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