Microbial cracking method and kit thereof

A microorganism and kit technology, applied in the field of microbial lysis methods and their kits, can solve the problems of missed pathogen detection, cell wall thickness, poor lysis effect, etc. The effect of wall efficiency

Pending Publication Date: 2021-08-10
深圳华大因源医药科技有限公司 +2
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  • Application Information

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Problems solved by technology

However, in the face of unknown microorganisms in clinical samples, and the cell walls of some microorganisms are covered with capsular layers, resulting in thicker cell walls; the cell walls of microorganisms such as filamentous fungi contain a large amount of chitin (also known as chitin, which is N-acetylglucosamine The structure formed by β-linkage polymerization is the same as polysaccharide), resulting in tough cell walls, and the existing extraction methods cannot effectively crack the cell walls, resulting in low efficiency of microbial nucleic acid extraction, which is prone to missed detection of pathogens
[0006] For the lysis of microorganisms for the purpose of DNA extraction, the lysis effect of existing methods is not good

Method used

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  • Microbial cracking method and kit thereof
  • Microbial cracking method and kit thereof
  • Microbial cracking method and kit thereof

Examples

Experimental program
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Embodiment 1

[0083] This embodiment uses the method of experimental group 1 and the method of not breaking the wall (experimental group 2), the separate enzymatic hydrolysis method (experimental group 3), the separate bead milling method (experimental group 4), the method of experimental group 5 (enzyme addition amount) respectively 4.5 μL), the method of experimental group 6 (enzyme addition amount is 12 μL), the method of experimental group 7 (glass bead diameter 1 mm), the method of experimental group 8 (glass bead volume 100 μL), the method of experimental group 9 (glass bead Volume 400μL) for parallel testing on the same sample. This embodiment uses simulated samples. In order to test the comprehensiveness of the detection process for microbial detection, when configuring the simulated samples, Staphylococcus aureus (G+), Staphylococcus hemolyticus (G+), Escherichia coli (G-), Pseudomonas aeruginosa sp. (G-), Klebsiella pneumoniae (G-), Stenotrophomonas maltophilia (G-), Candida albic...

Embodiment 2

[0100] The nucleic acid extraction reagent used in this example is the same as in Example 1.

[0101] In this example, Aspergillus fumigatus was cracked and tested. Aspergillus fumigatus (Latin name Aspergillus fumigatus) is an important pathogen, and pulmonary aspergillosis is mainly caused by Aspergillus fumigatus infection.

[0102]A case of alveolar lavage fluid sample (volume 450 μL) was taken for experiment, and the sample was obtained from human alveolar lavage with physiological saline (aqueous sodium chloride solution with a mass concentration of 0.9%). With reference to the method of Example 1, this embodiment uses the method of experimental group 1 and the non-broken treatment method (experimental group 2), the separate enzymatic hydrolysis method (experimental group 3), the separate bead milling method (experimental group 4), and the experimental group The method of 5 (enzyme addition amount is 4.5 μL), the method of experimental group 6 (enzyme addition amount is ...

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Abstract

The invention relates to a microbial cracking method and a kit thereof, and the microbial cracking method comprises the following steps: an enzymolysis step: cracking a microbial sample to be detected by using fungal cell wall lyase; and a physical cracking step: grinding and cracking the to-be-detected microbial sample obtained by cracking in the enzymolysis step by using mixture glass beads of cracking media with different particle sizes to obtain a cracking solution, namely the cracking solution for nucleic acid extraction. The enzymolysis method is combined with physical grinding and cracking, and cracking media with different particle sizes are used for physical cracking, so that the wall breaking efficiency is effectively improved, the sample detection sensitivity is effectively improved, and the method is suitable for wall breaking of microbial cells of different sample types and has high clinical universality.

Description

technical field [0001] The invention relates to the technical field of microbial genome detection, in particular to a microbial lysing method and a kit thereof. Background technique [0002] The existing microbial cell wall fragmentation methods mainly include physical methods and chemical methods. Physical methods mainly include ultrasonic method, glass bead grinding method, and liquid nitrogen freeze-thaw method; chemical methods mainly include biological enzymatic hydrolysis method and chemical reagent method. [0003] The ultrasonic method in the physical method is mainly based on the shear force of the ultrasonic wave on the cell wall to break the wall, but the effect on the wall breaking of filamentous fungi is not good, and the flux is small, which cannot meet the needs of the current sample size. The glass bead grinding method is widely used and can effectively lyse the cell wall, but different types of pathogens require different grinding conditions. The liquid ni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/06C12Q1/6806
CPCC12N1/06C12N1/066C12Q1/6806Y02A50/30
Inventor 罗伟伟申奥宫艳萍吴红龙
Owner 深圳华大因源医药科技有限公司
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