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Distinctive method for quantitatively detecting staphylococcal bacteria

A technique for the quantitative detection of staphylococci, which is applied in the field of specific quantitative detection of staphylococci, can solve problems such as economic losses, threats to food health, food contamination, etc., and achieve the effect of fast and simple operation and good application prospects

Active Publication Date: 2014-01-15
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Not only humans, but livestock are also susceptible to Staphylococcus infection. Staphylococcus infection can lead to mastitis in cows and infectious arthritis in chickens, which seriously threatens food health, leads to food contamination, and brings major economic losses.

Method used

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  • Distinctive method for quantitatively detecting staphylococcal bacteria
  • Distinctive method for quantitatively detecting staphylococcal bacteria
  • Distinctive method for quantitatively detecting staphylococcal bacteria

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0064] Preparation of LB liquid medium: Trypton1%, Yeast Extract0.5%, NaCl1%, pH7.0, autoclaved at 121°C for 20min;

[0065] When making LB solid plates, add 1.5% agar powder to the LB liquid medium, and autoclave at 121°C for 20 minutes;

[0066] When making LB semi-solid plates, add 0.75% agar powder to the LB liquid medium, and autoclave at 121°C for 20 minutes;

[0067] The above % means mass volume percentage, g / 100ml.

[0068] Restriction enzymes and T4 DNA ligase were purchased from NEB Company.

[0069] Nickel prepacked columns were purchased from Shanghai Sangon Bioengineering Co., Ltd.

[0070] Profile-13560 (10X) ATP microbial rapid detection system was purchased from Beijing Haozheng Zhixin Technology Co., Ltd.

[0071] The SRA reagent was purchased from Beijing Haozheng Zhixin Technology Co., Ltd., and the Profile-13560 (10X) ATP microbial rapid detection system came with it.

[0072] Luciferase was purchased from Beijing Haozheng Zhixin Technology Co., Ltd., ...

Embodiment 1

[0074] Embodiment 1, the acquisition of recombinant lysostaphin

[0075] 1. Construction of expression vector

[0076] (1) The gene sequence of Lysostaphin (Staphylococcus simulans by stashylolyticus) (Gene ID: 8864975) was obtained by BLAST on NCBI, and the gene sequence was optimized to synthesize the sequence shown in SEQ ID No.1, wherein Lysostaphin The gene of the enzyme is the DNA molecule shown in the 37th to the 777th nucleotide from the 5' end in SEQ ID No.1, and the amino acid sequence of lysostaphin is the 13th from the N-terminus in SEQ ID No.2 from position 258 to amino acid 258. The optimized sequence makes it easier for lysostaphin to be soluble expressed in the Escherichia coli prokaryotic expression system.

[0077] (2) Digest the DNA molecule shown in SEQ ID No.1 with BamHI and HindIII to obtain a gene fragment; use BamHI and HindIII to double digest the expression vector pQE30 to obtain a large fragment of the vector; connect the gene fragment to the large...

Embodiment 2

[0106] Embodiment 2, drop method detects recombinant lysostaphin bactericidal activity in vitro

[0107] One strain of Staphylococcus aureus and 173 strains of clinically isolated Staphylococcus aureus were cultured to the logarithmic phase, and 500ul each was taken to spread double-layer LB plates, and stood at room temperature for 10min. Pipette 1ul of recombinant lysostaphin droplet on the plate, and use 0.01mol / L PBS with pH7.2 as blank control, and each group has three parallels. Incubate the plate upside down at 37°C for 3-4 hours, observe the results, and the results are as follows image 3 shown.

[0108] image 3 A is the recombinant lysostaphin group, image 3 B is the PBS (blank control) group.

[0109] image 3 It showed that there were clear circular lysis spots in the area dripped with recombinant lysostaphin, but there was no lysis spots in the PBS (blank control) group, indicating that the recombinant lysostaphin had bactericidal activity against Staphyloc...

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Abstract

The invention discloses a distinctive method for quantitatively detecting staphylococcal bacteria. The invention discloses recombinant lysostaphin shown as the following (1) or (2): (1) a protein shown as N-terminal amino acids (13-258 positions) in SEQ ID No. 2; (2) a protein formed by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence (1) and having the same function. The distinctive method for quantitatively detecting staphylococcal bacteria, disclosed by the invention, is recombinant lysostaphin-integrated ATP bioluminescence method. The method disclosed by the invention is rapid and easy to operate, provides a theoretical support for on-site or clinical quantitative detection of staphylococcal bacteria, and has a good application prospect.

Description

technical field [0001] The invention relates to a method for specific and quantitative detection of staphylococcus. Background technique [0002] Staphylococcus is a common flora on the surface of human skin, but it is also a very important pathogenic bacteria that can cause life-threatening diseases ranging from skin diseases to pneumonia and bacteremia. Not only humans, but livestock are also susceptible to Staphylococcus infection. Staphylococcus infection can lead to mastitis in cows and infectious arthritis in chickens, which seriously threatens food health, leads to food contamination, and brings major economic losses. Notably, the increasing infection of drug-resistant Staphylococcus aureus, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA), has become a major concern because golden yellow Staphylococci are the most common cause of nosocomial infections. Therefore, it is very necessary to establish a met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/52C12N15/57C12N15/70C12N1/21C12Q1/37C12Q1/14C12R1/445
CPCC12N9/52C12Q1/14C12Q1/37C12Y304/24075
Inventor 童贻刚李玉元米志强安小平
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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