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Gene chip for detecting important drug resistant gene of pseudomonas aeruginosa and kit thereof

A technology of Pseudomonas aeruginosa and drug-resistant genes, which is applied in the direction of microbial-based methods, microbial measurement/testing, chemical libraries, etc., can solve the problem of high chip manufacturing cost, achieve low cost, cost reduction, and good stability Effect

Inactive Publication Date: 2011-02-09
TIANJIN BIOCHIP TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cost of the chip is relatively high, and the detection cost of each sample is about 50 US dollars. Only one probe is designed for each target gene, which is prone to false positives
[0008] At present, there are no literature reports or invention patents on the detection chip for the drug resistance gene of Pseudomonas aeruginosa in China

Method used

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  • Gene chip for detecting important drug resistant gene of pseudomonas aeruginosa and kit thereof
  • Gene chip for detecting important drug resistant gene of pseudomonas aeruginosa and kit thereof
  • Gene chip for detecting important drug resistant gene of pseudomonas aeruginosa and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Probe Design and Preparation

[0050] 1. Sequence to get:

[0051] The entire gene sequence of the above-mentioned drug resistance genes of Pseudomonas aeruginosa and the typical sequence of bacterial 16S rRNA gene were downloaded from the GenBank public database.

[0052] 2. Probe design:

[0053] Clustal X software was used to compare the different sequences of each gene. According to the comparison results, a representative sequence was selected and imported into Primer Premier 5 software. Run the program to design the probe, select the length (25 ± 5) bp, T m 60 ℃ ~ 70 ℃, GC content 40% ~ 60%, the designed probe was imported into NCBI for blastn comparison to ensure the intragene conservation and intergene specificity of the sequence. At least 4 different probes were designed for each gene for subsequent screening.

[0054] 3. Probe synthesis: The designed probe sequence is added to the total length of 40 nucleotides (positive control probe is lengt...

Embodiment 2

[0061] Embodiment 2 Primer design and preparation

[0062] 1. Examples of PCR primer design:

[0063] (1) Example of the design of universal amplification primers for a single drug resistance gene TEM: Import all TEM sequences of Pseudomonas aeruginosa (including each TEM genotype) into Glustal X software, and select a representative sequence from it and import it into Primer Primer 5.0 In the software, the length is set to (20±4) bp, the G+C% value is 40% to 60%, and the product size is greater than 200 bp. And find the nucleotide sequence region suitable for the design of universal primers, and its characteristics must meet the following conditions: 1, the constant region should include most TEM types; 2, the region should contain a region that is easy to design specific probes 3. The constant regions on both sides of this region can meet the design of primers. The primer sequence with the smallest secondary structure (hairpin structure, dimer) was selected, and the sequ...

Embodiment 3

[0069] Embodiment 3 Detection of 10 common important drug resistance genes of Pseudomonas aeruginosa by gene chip and preparation of the kit

[0070] 1. Pre-hybridization of the chip

[0071] (1) Put the ordered chip into 500 ml of pre-hybridization solution preheated at 42°C, and incubate at 42°C for 1 hour.

[0072] Pre-hybridization solution formula: 1% BSA (bovine serum albumin)

[0073] 5×SSC (sodium chloride-sodium citrate solution)

[0074] 0.1% SDS (sodium dodecyl sulfate)

[0075] (2) at ddH 2 Washed in O medium twice for 1 minute, dehydrated in 95% ethanol for 2 minutes, dried with a hair dryer, and set aside.

[0076] 2. Obtaining total DNA:

[0077] (1) The collected Pseudomonas aeruginosa was picked up and the monoclonal colony was transferred into 5ml of 2YT culture solution for 37°C shaking culture for 12 hours;

[0078] (2) Take 1ml of bacterial liquid into a 1.5ml centrifuge tube, centrifuge at 15000g × room temperature for 3 minutes;

[0079] (3) Dis...

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Abstract

The invention provides a gene chip for detecting an important drug resistant gene of pseudomonas aeruginosa and a kit thereof. The gene chip comprises a solid-phase carrier and an oligonucleotide probe fixed on the solid-phase carrier, wherein the probe comprises a DNA fragment or a complementary DNA fragment thereof selected from carb, tem, imp, oxa group I, oxa group II, aac (6')-Ib, aac (6')-II, aac (3')-I, aac (3')-II, aadB and a bacterium 16S rRNA gene. The gene chip and the detecting kit can achieve the aim of simultaneously detecting the 10 drug resistant genes of the pseudomonas aeruginosa, have the characteristics of high pertinence, high specificity, high sensitivity, high detecting speed, high repeatability, and the like and are suitable for a hospital, a disease preventing control mechanism and other clinical laboratories to carry out drug resistant gene detection and drug resistance monitoring on the pseudomonas aeruginosa.

Description

technical field [0001] The invention relates to a gene chip and a detection kit comprising the same, in particular to a gene chip and a detection kit for detecting an important drug resistance gene of Pseudomonas aeruginosa. Background technique [0002] Pseudomonas aeruginosa (Pseudomonas aeruginosa) is a Gram-negative non-fermentative opportunistic pathogen. It is easy to grow and reproduce in the outside world. It is widely distributed, has strong drug resistance, and has various transmission routes. It has become one of the most common pathogens of nosocomial infections today. . 10% of infections in the intensive care unit are related to Pseudomonas aeruginosa, and pneumonia and sepsis caused by Pseudomonas aeruginosa show high morbidity and mortality. In recent years, the resistance rate of Pseudomonas aeruginosa to antibiotics has increased year by year, and the detection rate of multidrug-resistant Pseudomonas aeruginosa has also increased. [0003] Pseudomonas aeru...

Claims

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Application Information

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IPC IPC(8): C40B40/06C12Q1/68C12R1/385
Inventor 王磊康海蔚曹勃阳冯露
Owner TIANJIN BIOCHIP TECH CO LTD
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