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Method for extracting nucleic acid of gram positive bacteria
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A gram-positive and wall-gram-positive technology is applied in the field of nucleic acid extraction of gram-positive bacteria, which can solve the problems of high price, unsatisfactory extraction effect, complicated operation steps, etc., and achieves low cost, good extraction effect, Simple to use effects
Active Publication Date: 2014-06-18
CAPITALBIO CORP +1
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These kits are expensive, the operation steps are cumbersome, and the extraction effect is not ideal
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Embodiment 1
[0080] The nucleic acid extraction of embodiment 1, mycobacterium
[0081] Mycobacterium is a typical Gram-positive bacteria, which contains a variety of highly harmful pathogenic bacteria, such as Mycobacterium tuberculosis, Mycobacterium leprae, etc. The nucleic acid extraction of such bacteria plays an important role in the molecular diagnosis of pathogens. significance. Mycobacterium smegmatis with lower risk is selected as a model to verify the nucleic acid extraction effect of the nucleic acid extraction method of the present invention in Gram-positive bacteria-Mycobacterium smegmatis.
[0082] Strain: Mycobacterium smegmatis (Latin name Mycobacterium smegmatis, strain purchased from ATCC, USA, strain number 19420,)
[0083] Reagents: extract A, extract B, chloroform, absolute ethanol, 75% ethanol, RNase A (7000units / ml), binding column (purchased from MN company, catalog number 740903), binding solution (concentration of 4.6M Guanidine isothiocyanate aqueous solution)...
Embodiment 2
[0140] Embodiment 2, the nucleic acid extraction of Staphylococcus aureus
[0141] So far, in human bacterial infection cases, the infection rate of Gram-positive bacteria can account for 30-40% of all bacterial infections, among which Staphylococcus aureus is one of the main pathogens. Therefore, Staphylococcus aureus is a very representative Gram-positive bacterial pathogen. The purpose of this experiment is to verify the nucleic acid extraction effect of the method of the present invention in Gram-positive bacteria-Staphylococcus aureus.
[0142] Strain: Staphylococcus aureus (Latin name Staphyloccocus aureus, strain number ATCC25923.)
[0144] Medium: Brain Heart Infusion (BHI) (sold by the company OXOID, catalog number CM1135)
[0145] 1. Extract...
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Abstract
The invention discloses a method for extracting nucleic acid of gram positive bacteria. The invention provides a reagent for breaking cell walls of gram positive bacteria, wherein the reagent comprises extract A and extract B; the extract A is prepared according to the following method of mixing Tris, EDTA (Ethylene Diamine Tetraacetic Acid), SDS (Sodium Dodecyl Sulfate), Tween 80, Triton X-100, Brij58, 3-[(3-cholesterol aminopropyl) dimethylamino]-1-propanesulfonic acid, sarcosyl, beta-mercaptoethanol and water; and the extract B is prepared by the following method of mixing water phenol, ethanol and pyrrolidone. Experiments prove that the method for extracting the nucleic acid of gram positive bacteria is efficient, is simple in operation, low in cost, good in extraction effect, particularly good in RNA (Ribose Nucleic Acid) extraction effect and superior to the commercially available kits which are widely used at present.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a method for extracting Gram-positive bacterial nucleic acid. Background technique [0002] Bacteria are the pathogens that cause most infectious diseases, and the pathogenic bacteria that cause clinical bacterial infections are divided into two categories: Gram-positive bacteria and Gram-negative bacteria. So far, although gram-negative bacteria still occupy a dominant position in the bacterial spectrum of human infection, the infection rate of gram-positive bacteria has a tendency to increase year by year, and now it can account for 30-40% of all bacterial infections , of which 95% were coagulase-negative gluconococci, Staphylococcus aureus and enterococci. Whether it is a molecular diagnostic technology for the detection of pathogenic microorganisms or a transcriptome research technology for bacterial molecular biology research, the first step is the preparation of template nucle...
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