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Use of tail fiber protein of pseudomonas aeruginosa phage for preparing bacteria detection reagent

A technology of Pseudomonas aeruginosa and tail filament protein, which is applied in the direction of phage, virus/phage, biological testing, etc., can solve the problems of shortening detection time, effort, cross-immunogenicity of antibody batch differences, etc., and achieve rapid detection Effect

Pending Publication Date: 2019-01-04
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As the "gold standard", the traditional culture method is accurate and sensitive, but time-consuming and laborious; the PCR method based on the genetic material of pathogenic bacteria can significantly shorten the detection time and has high sensitivity, but PCR requires skilled operation skills and nucleic acid extraction steps , and under harsh environmental conditions, the activity of the enzyme will be inhibited; the immunoassay method is based on the combination of the antigenic component of the pathogenic bacteria and the antibody, which has the characteristics of rapidity and high specificity, but the antibody has batch differences and cross-immunogenicity

Method used

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  • Use of tail fiber protein of pseudomonas aeruginosa phage for preparing bacteria detection reagent
  • Use of tail fiber protein of pseudomonas aeruginosa phage for preparing bacteria detection reagent
  • Use of tail fiber protein of pseudomonas aeruginosa phage for preparing bacteria detection reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Gene recombinant expression of Pseudomonas aeruginosa phage tail filament protein P069

[0041] 1. Materials

[0042] Pseudomonas aeruginosa phage, the strain name is PAP1, and the preservation number is CGMCC No.15569.

[0043] Vector pET-21a (Novagen)

[0044] 2. Method

[0045] 1. Extraction of target gene of Pseudomonas aeruginosa phage tail filament protein P069

[0046] (1) Add 20 μL of 10 μg / μL RNase A and 30 μL of 5 μg / μL DNase I to 950 μL of phage PAP1, and let stand at 37°C for 1 hour;

[0047] (2) Add 1.0mL 40mM EDTA (pH 8.0) to inactivate DNase I;

[0048] (3) Add 5.0 μL of proteinase K and 10 mg of SDS, mix well, and incubate at 56°C for 1 hour to lyse the phage capsid, release DNA, and degrade residual DNase I;

[0049] (4) Add 2.0 mL of balanced phenol (pH 8.0), centrifuge at 5000 g and 4°C for 10 min, and collect the aqueous layer;

[0050] (5) Add 2.0 mL of chloroform, centrifuge at 5000 g and 4°C for 10 min, and collect the aqueous lay...

Embodiment 2

[0073] Example 2. Bioluminescent detection of Pseudomonas aeruginosa based on single site recognition of Pseudomonas aeruginosa phage tail filament protein P069

[0074] 1. Materials

[0075] Pseudomonas aeruginosa phage tail filament protein P069 was prepared according to the method in Example 1.

[0076] Pseudomonas aeruginosa phage, the strain name is PAP1, and the preservation number is CGMCC No.15569.

[0077] Magnetic nanoparticles (Lab on a bead LOABeadsTM AffiAmino, Sweden)

[0078] 2. Method

[0079] 1. Preparation of detection reagents

[0080] (1) Magnetic nanoparticles functionalized with phage tail filament protein P069:

[0081] Take 1.0mL of magnetic particles, wash with 1.0mL of PBST twice; add 1.0mL of PBST and 50μL of activation buffer, react for 15min, then wash with 1.0mL of PBST once, add 1.0mL of 1.0mg / mL React P069 solution at room temperature for 1 hour, then wash twice with PBST, add 80 μL of blocking agent, react at room temperature for 45 minute...

Embodiment 3

[0098] Example 3. Sandwich fluorescence detection of Pseudomonas aeruginosa based on two-site recognition of Pseudomonas aeruginosa phage tail filament protein P069

[0099] 1. Materials

[0100] Pseudomonas aeruginosa phage tail filament protein P069 was prepared according to the method in Example 1.

[0101] Pseudomonas aeruginosa phage, the strain name is PAP1, and the preservation number is CGMCC No.15569.

[0102] DMSO: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0103] TRITC: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0104] 2. Method

[0105] 1. Preparation of detection reagents

[0106] (1) Phage tail filament protein P069: prepared according to the method in Example 1

[0107] (2) Pseudomonas aeruginosa bacterial suspension standard substance:

[0108] Take a single colony of Pseudomonas aeruginosa and inoculate it in LB medium, culture it with shaking at 37°C for 5.5 hours, resuspend the bacteria with Tris buffer (...

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Abstract

The invention relates to use of a tail fiber protein of pseudomonas aeruginosa phage for preparing a bacteria detection reagent, and belongs to the technical field of microbiological detection. The preservation number of the pseudomonas aeruginosa phage is CGMCC No. 15569. The tail fiber protein P069 of the pseudomonas aeruginosa phage is used for preparing the bacteria detection reagent. The usedisclosed by the invention has the beneficial effects that the tail fiber protein P069 capable of specifically identifying pseudomonas aeruginosa and is used as a molecular identification reagent to specifically capture the pseudomonas aeruginosa, and other analysis technologies are combined to detect whole cells of the pseudomonas aeruginosa. The molecular identification reagent has the advantages of quick detection, convenience, accuracy, specificity, sensitivity and stability, is expected to being applied to site detection and quick screening of the pseudomonas aeruginosa, and can provide apowerful technological supporting platform for detecting the pseudomonas aeruginosa in the field of clinical diagnosis, food safety, environmental detection and the like.

Description

technical field [0001] The invention relates to the use of Pseudomonas aeruginosa phage tail silk protein for preparing bacteria detection reagents, belongs to the technical field of microbial detection, and especially refers to the use of Pseudomonas aeruginosa phage tail silk protein P069 as a molecular recognition reagent for Pseudomonas aeruginosa , a technology that applies it to the detection of Pseudomonas aeruginosa. Background technique [0002] Pseudomonas aeruginosa (Pseudomonas aeruginosa) is a fermented Gram-negative pathogenic bacteria, one of the three opportunistic pathogens, and has a high fatality rate in patients with pulmonary cystic fibrosis. leading cause of infection in pediatric intensive care. Therefore, establishing a rapid, convenient and sensitive detection method for Pseudomonas aeruginosa is of great significance for human health and disease treatment. [0003] The existing detection methods for Pseudomonas aeruginosa mainly include traditiona...

Claims

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Application Information

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IPC IPC(8): C12N7/00C07K14/005C12Q1/70C12Q1/04G01N33/68G01N33/569C12R1/385
CPCC07K14/005C12N7/00C12N2795/00021C12N2795/00022C12Q1/04C12Q1/70G01N33/56911G01N33/68G01N2333/21
Inventor 付志锋何勇卢曙光
Owner SOUTHWEST UNIVERSITY
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