Medicine and bacterium resistant detection chip, method for preparation and application thereof

A technology for detecting chips and drug-resistant bacteria, applied in the field of microbial testing, can solve problems such as difficulty in popularization and application, high operational requirements, and poor practicability.

Inactive Publication Date: 2005-07-06
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such as nucleic acid hybridization technology, target gene amplification technology, etc., but the common disadvantage of these technologies is that there are few target sites that can be detected, and the practicability is poor. Some technologies require RNA manipulation, which is complicated and requires high operation. It is difficult to promote the application on the

Method used

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  • Medicine and bacterium resistant detection chip, method for preparation and application thereof
  • Medicine and bacterium resistant detection chip, method for preparation and application thereof
  • Medicine and bacterium resistant detection chip, method for preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Embodiment 1 Probe Design and Chip Fabrication

[0111] According to the known 23S rRNA gene sequences and drug resistance gene sequences of different bacteria, design probes with similar length, about 20mer. As shown in Table 1 and Table 2.

[0112] The synthesized oligonucleotide probes were dissolved in ionized water and mixed with equal volumes of the spotting solution so that the final concentration was 75 pmol / μl; The surface of the glass slide modified by aldehyde groups; placed in a relative humidity of 70%, fixed at room temperature for 48 to 72 hours; then, at room temperature, immerse the glass slide in 0.2% SDS and shake for several minutes, then immerse in double distilled water Shake for a few minutes, then immerse in 100°C double distilled water for 30 seconds, dry and set aside.

Embodiment 2

[0113] Example 2 Fluorescent labeling of target DNA fragments in specimens

[0114] Design two pairs of primers, and label the 5' end of each pair of primers with Cy3 or Cy5, and use PCR to amplify the target fragment of fluorescently labeled sample DNA, where the template is bacterial genomic DNA, 23s RNA gene primer sequence and drug resistance gene The primer sequences are listed in Table 3. The amplification system is as follows: Mix 2 μl of sample DNA solution with 23 μl of PCR amplification system (the amplification system includes 2.5 μl of 10×PCR reaction buffer, 5 nmol dATP, 5 nmol dGTP, 5 nmol dGTP, 5 nmol dCTP, and 15 pmol upstream and downstream primers each) . The system was kept at 95°C for 5min, then cycled 40 times at 95°C for 30S, 55°C for 30S, and 72°C for 30S, and finally kept at 72°C for 5min.

Embodiment 3

[0115] Example 3 Hybridization of the labeled fragment of interest with the chip

[0116] Take 2 μl of the target DNA fragment sample containing the fluorescently labeled specimen, mix it with 13 μl of Easy Hyb hybridization solution (Roche), denature at 99°C for 5 minutes, take 15 μl and drop it on the surface of the chip after ice bathing, cover the cover glass, and place it in a 40°C humid chamber Hybridization in medium for 60 min, then rinsed in washing solution I (1×SSC, 0.1% SDS) at room temperature in the dark for 10 min, and dried in the air.

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Abstract

The invention relates to a detection chip for familiar resistant organisms and its preparation and application methods belonging to microorganism detection field. The invention employs DAN chip method including fixing the synthesized oligonucleotide probes for clinical detection of familiar resistant organisms on the slide surface to form a dot-plot, hybridizing the pending sample DNA with the chip to obtain a great deal of gene sequence information relative to the bacteria identification and drug tolerance, identifying the kinds of the familiar pathogenicbacterias existing in the clinical sample, thus to implement the identification of the clinical familiar pathogenicbacterias and detection of the main drug resistant spectrum. The invention is simple, convenient, quick, sensitive and specific, and can obviously shorten the disease diagnosis time.

Description

technical field [0001] The invention belongs to the field of microbial inspection, and relates to a method for rapid detection of drug-resistant bacteria, in particular to a detection chip for common drug-resistant bacteria and its preparation method and application method. Background technique [0002] The DNA chip detection technology developed in recent years can quickly and efficiently acquire or process a large amount of life information (including gene identification, gene mutation detection, gene expression profile detection) by using or relying on the intensive and parallel processing principles of microelectronic chip technology. and identification of foreign molecules, etc.), has played an important role in biological research such as life sciences, medical diagnosis, new drug screening and forensic identification. [0003] At present, infectious diseases are still important diseases that threaten human health and life. According to the World Health Organization He...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 林东昉徐晓刚毛红菊赵建龙赵辉朱德妹张婴元汪复
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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