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Probes and gene chip for pseudomonas aeruginosa nucleic acid detection, and kit

A Pseudomonas aeruginosa and detection kit technology, applied in the field of molecular biology, can solve problems such as weak signal, achieve the effects of simplifying testing steps, reducing investment costs and application costs, and widening the potential of the clinical application market

Inactive Publication Date: 2018-05-18
重庆威斯腾生物医药科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its signal is weak and must be excited to emit light, requiring expensive laser scanning equipment

Method used

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  • Probes and gene chip for pseudomonas aeruginosa nucleic acid detection, and kit
  • Probes and gene chip for pseudomonas aeruginosa nucleic acid detection, and kit
  • Probes and gene chip for pseudomonas aeruginosa nucleic acid detection, and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] [Example 1] Design of target fragments, primers and probes

[0027] The gene sequence of Pseudomonas aeruginosa was searched through the NCBI database, and the conserved sequences of PAK gene and Pf4 gene were selected as targets (IDs were CP020659 and KM975472, respectively). Probes, in order to make the chip results can be read directly by naked eyes after being developed with the chromogenic solution, the 5' ends of the upstream and downstream primers of each pathogen target gene are labeled with biotin. The specific sequence is as follows:

[0028] PAK gene

[0029] PAK gene target sequence amplification primers

[0030] Primer F (PAK-F): the sequence is SEQ ID NO.5;

[0031] R primer (PAK-R): the sequence is SEQ ID NO.6;

[0032] PAK probe 1: the sequence is SEQ ID NO.1;

[0033] PAK probe 2: the sequence is SEQ ID NO.2;

[0034] Pf4 gene

[0035] F primer (Pf4-F): the sequence is SEQ ID NO.7;

[0036] R primer (Pf4-R): the sequence is SEQ ID NO.8;

[0037]...

Embodiment 2

[0039] [Example 2] Preparation of chip

[0040] Amino-modified probes and control probes were arranged according to Table 1, and the probes were respectively spotted on the NC. Two parallel spotting points were set for each group, and the probes were immobilized by ultraviolet crosslinking for 20 minutes.

[0041] The sequence of the control ProbeT probe is SEQ ID NO.11.

[0042] Table 1 Pseudomonas aeruginosa microarray matrix

[0043]

Embodiment 3

[0044] [Example 3] Detection method

[0045] 1. Genome extraction: This kit does not provide bacterial genomic DNA extraction reagents, use the bacterial DNA extraction kit from Bao Bio Engineering (Dalian) Co., Ltd., product number 9763.

[0046] 2. Amplification system, the components are shown in Table 2.

[0047] Table 2 PAK, Pf4 gene multiplex PCR amplification system

[0048]

[0049] The PCR reaction program was: 95°C for 5min; 32 cycles of 95°C for 10s, 58°C for 30s, 72°C for 30s; 72°C for 10min.

[0050] 3. Hybridization

[0051] Materials: The negative control substance is double distilled water; the positive control is the target sequence amplified using the PAK and Pf4 genes as templates and the plasmid constructed by the 19T vector; the hybridization buffer is Quick Reaction Buffer (provided by Chongqing Weisten Biological Co., Ltd.), The cleaning solution formula is to dissolve 8g NaCl, 0.2g KCl and 3g Tris-base in 800ml distilled water. Adjust the pH to 7...

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Abstract

The invention relates to probes and a gene chip for pseudomonas aeruginosa nucleic acid detection, and a kit. The invention discloses a group of probes for pseudomonas aeruginosa detection, includingbacterial PAK gene probes 1 and 2 of which the sequences are respectively SEQ ID NO.1 and 2 as well as bacterial Pf4 gene probes 1 and 2 of which the sequences are respectively SEQ ID NO.3 and 4. Theinvention also discloses the gene chip containing the probes and the kit of the gene chip. By optically amplifying hybridization signals through visible light, the signals have a high resolution and can be directly identified. The probes, the gene chip and the kit disclosed by the invention have the advantages that the operation is simple, the manufacturing cost is low, the results are easy to read, on-site quick detection requirements of hospital clinics are met, and the results are accurate and reliable.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a probe, a gene chip and a kit for nucleic acid detection of Pseudomonas aeruginosa. Background technique [0002] Pseudomonas aeruginosa (Pseudomonas aeruginosa), also known as Pseudomonas aeruginosa, is an opportunistic pathogen and one of the main pathogens of nosocomial infections. It often causes postoperative wound infection, and can also cause bedsores, abscesses, and suppurative otitis media. Pseudomonas aeruginosa infection can occur in many anatomical sites, including skin, subcutaneous tissue, bone, ear, eye, urinary tract, and heart valves. The site of infection is related to the entrance of bacteria and the susceptibility of the patient. In burns, the area under the eschar can become the place where a large number of bacteria invade, and then become the focus of causing bacteremia, and bacteremia is often a fatal complication of burns. Pseudomonas aeruginosa, which ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C40B40/08C12R1/385
CPCC12Q1/689C40B40/08
Inventor 周勇项周
Owner 重庆威斯腾生物医药科技有限责任公司
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