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Gene chip for detecting 11 types of common infectious diarrheal disease pathogen and application thereof

An infectious diarrhea and gene chip technology, applied in the field of gene chips, can solve the problems of high similarity and inability to design probes, and achieve the effects of strong specificity, good sensitivity and high detection throughput

Inactive Publication Date: 2014-07-09
YANTAI YUHUANGDING HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Because the 16s rRNA and gyrB genes of hemorrhagic Escherichia coli O157:H7 and Shigella are very similar to the gene sequences of common Escherichia coli, it is impossible to design probes based on these two gene sequences

Method used

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  • Gene chip for detecting 11 types of common infectious diarrheal disease pathogen and application thereof
  • Gene chip for detecting 11 types of common infectious diarrheal disease pathogen and application thereof
  • Gene chip for detecting 11 types of common infectious diarrheal disease pathogen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 gene chip detection probe

[0055] 1. PCR amplification and electrophoresis detection

[0056] Perform PCR amplification and electrophoresis detection on the 16S rRNA gene of sample bacteria (Vibrio vulnificus, Shigella dysenteriae, Escherichia coli O157:H7, Salmonella, see Table 1.1), and the primer sequence is shown in SEQ ID NO: As shown in 118-119, the amplified electrophoresis results are as follows Figure 14 As shown, the sequencing results of the amplified products are shown in Table 1.2, and some screenshots of the sequencing peaks are shown in Figure 13 shown.

[0057] Table 1. Annotation of electropherogram after 116S rRNA gene PCR

[0058]

[0059] Table 1.2 Sequencing test results

[0060]

[0061] 2. Probe Design

[0062] For the microorganisms provided in the diarrhea project, a probe with a length of 40mer was designed to distinguish the target microorganisms. The microorganisms provided in the project are shown in Table 2.1 belo...

Embodiment 2

[0079] Example 2 gene chip

[0080] The chip manufacturing process of the diarrhea pathogenic bacteria gene chip includes: preparation of spotting probes, preparation of aldehyde-based substrates, chip printing, fixing and scanning, chip packaging and other steps. For the process route, see Figure 15 .

[0081] 1. Preparation of sampling probe

[0082] use Gene chip spotting solution and water, prepare the oligonucleotide probe into a specific concentration spotting solution, and then transfer it to the corresponding position of the 384-well plate according to the dot array pattern, and seal it with parafilm, ready for spotting.

[0083] 1.1 Selection of sampling solution

[0084] used in the preparation of gene chips for diarrhea pathogens The gene chip spotting solution is a product independently developed and commercialized by Boao Biotechnology Co., Ltd. (catalogue number: 440010). Comparison of the results after spotting with the gene chip spotting solution and u...

Embodiment 3

[0103] Embodiment 3 uses gene chip to carry out sample detection

[0104] 1. Sample collection and processing

[0105] The pathogenic microorganisms detected were enterohaemorrhagic Escherichia coli (E.coli), Shigella dysenteriae (S.dysenteriae), Salmonella (Salmonella), Vibrio vulnificus (V.vulnificus), Vibrio parahaemolyticus (V .parahaemolyticus), Vibrio cholerae (V.cholerae), Proteus vulgaris (P.vulgaris), Vibrio alginolyticus (V.alginolyticus), Vibrio furnissii (V.furnissii), Aeromonas hydrophila (A.hydrophila), Campylobacter jejuni (Campylobacter); the DNA of the sample was extracted from the above-mentioned microbial samples by a bacterial genome nucleic acid extraction kit (purchased from QIAGEN).

[0106] 2. Amplification and labeling of sample nucleic acids

[0107] 2.1 Polymerase chain reaction

[0108] 2.1.1 Universal amplification primers

[0109] Design and screen a universal primer pair that can amplify the full length of the target gene as broadly as possib...

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Abstract

The invention relates to a gene chip and an application, which belongs to the biological detection field. The invention designs a set of detection probes by aiming at 11 types of common infectious diarrheal disease pathogen microorganism in clinic, and the gene chip containing the set of the probe. The detection probes comprises a vibrio parahaemolyticus probe, a vibrio vulnificus, a vibrio cholera probe, a vibrio alginolyticus probe, a vibrio furnissii probe, a shigella probe, an escherichia coli probe, an aeromonas probe, a salmonella probe, a campylobacteria probe, and a proteusbacillus vulgaris probe; the above mentioned probe sequence is shown as SEQ ID No: 1-117. The gene chip and probe can detect 11 types of common infectious diarrheal disease pathogen, the detection flux is high, the specialty is strong, the sensitivity is high, and the detection is rapid and effective.

Description

technical field [0001] The invention relates to a gene chip, in particular to a gene chip for distinguishing and detecting 11 common clinical infectious diarrhea pathogenic bacteria, and belongs to the field of biological detection. Background technique [0002] Diarrhea is a gastrointestinal infectious disease with a high incidence and is prevalent all over the world. Its incidence is second only to upper respiratory tract infection. In my country, the incidence of infectious diarrhea ranks first among all infectious diseases. Infectious diarrhea is a common and frequently-occurring disease in my country. The etiology is relatively complex and can be caused by viruses, bacteria and protozoa. A rapid and efficient diagnostic method for common diarrhea-causing bacteria. [0003] At present, the traditional detection methods for pathogenic bacteria in diarrhea mainly include the following: (1) routine stool examination; (2) pathogenic examination; (3) serological examination; ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6837C12Q2531/113C12Q2565/501Y02A50/30
Inventor 孙成铭栾材富刘杰伊茂礼李少君吴金英张守信段丽君孙隽龚磊张成林
Owner YANTAI YUHUANGDING HOSPITAL
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