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Gene chip for detecting 15 clinical common pathogenic microorganisms

A technology of pathogenic microorganisms and gene chips, which is applied in the field of gene chips for detection of common clinical pathogenic microorganisms, can solve the problems of single probes, retention, and inability to fully meet the accurate detection of target microorganisms, etc.

Active Publication Date: 2013-03-06
GUANGZHOU IMPROVE MEDICAL TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, domestic microbial detection chips are mostly at the level of scientific research, and the probes are mostly single or only involve part of the 16SrRNA gene, which cannot fully meet the requirements for accurate detection of target microorganisms (Xue Jianya, Weng Xinhua et al., Construction of bacterial 16SrRNA gene chip and its application in Application in Bacteria Identification, Journal of Second Military Medical University, 2007, 28(8): 919-921)

Method used

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  • Gene chip for detecting 15 clinical common pathogenic microorganisms
  • Gene chip for detecting 15 clinical common pathogenic microorganisms
  • Gene chip for detecting 15 clinical common pathogenic microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] The preparation of embodiment 1 gene chip

[0092] In this example, an aldehyde-based glass slide is used as a solid phase carrier to illustrate the preparation method of the gene chip of the present invention. (1) Using aldehyde-treated glass slides as carriers;

[0093] (2) Design primers and probes:

[0094] Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli, Shigella, Enterobacter aerogenes, Pseudomonas fluorescens, Candida albicans, smooth Candida, Candida tropicalis, Candida parapsilosis, Aspergillus terreus, Aspergillus flavus, Rhizopus oryzae 16SrRNA gene and ITS gene sequence, through DNAMAN 5.2.2 (Lynnon Biosoft, USA) and NCBIBLAST software ( http: / / www.ncbi.nlm.nih.gov / BLAST / ) to find their conserved regions and hypervariable regions, and then use Primer Premier 5.0 (PREMIERBiosoft International, CA) to design primers in these conserved regions, Design probes in hypervariable regions. After trial an...

Embodiment 2

[0112] The detection of embodiment 2 pathogenic microorganisms

[0113] (1) Extract bacterial DNA

[0114] The DNA of pathogenic microorganisms in Table 4 was respectively extracted by CTAB / NaCl method as templates.

[0115] Table 4

[0116]

[0117] 1: ATCC: American Type Culture Collection

[0118] 2: CCTCC: China National Type Culture Collection Center

[0119] (2) target DNA amplification

[0120] The 4 pairs of primers in Tables 1 and 2 were used for PCR reaction to amplify the target DNA. The multiplex PCR reaction solution consists of 10×buffer (Biostar), 10 μmol / L of each primer, 10mmol / L dNTPs (Roche), 2U Taq DNA polymerase (Biostar), Cy3-dCTP (GE) and sterile water. The PCR reaction conditions are:

[0121] 50μl system (multiplex PCR):

[0122] Taq buffer (10×) 5μl

[0123] dNTPs (10mM) 0.8μl

[0124] srr-rl 1.5 μl

[0125] srr-dg 1.5 μl

[0126] srr-ra 1.5 μl

[0127] srr-pg 1.5μl

[0128] srr-gd 1.5 μl

[0129] srr-ak 1.5μl

[0130] Taq (2U / μl) ...

Embodiment 3

[0152] The detection of embodiment 3 pathogenic microorganisms

[0153]In this example, according to the method of Example 2, 15 strains were cultured and biochemically identified as Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli, Shigella, Enterobacter aerogenes, Clinical isolation of Pseudomonas fluorescens, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Aspergillus terreus, Aspergillus flavus and Rhizopus oryzae (Department of Laboratory, Zhongnan Hospital of Wuhan University, Wuhan, Hubei ) for the detection of pathogenic microorganisms. The results showed that the detection results were completely consistent with the results of culture and biochemical identification.

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Abstract

The invention discloses a gene chip for detecting 15 clinical common pathogenic microorganisms. The gene chip comprises specific detection probes fixed on a solid phase vector, wherein the probes are designed for 16SrRNA gene and ITS gene sequences of staphylococcus aureus, klebsiella pneumoniae, pseudomonas aeruginosa, proteus mirabilis, Escherichia coli, shigella, enterobacter aerogenes, pseudomonas fluorescens, candida albicans, candida glabrata, candida tropicalis, candida parapsilosis, aspergillus terreus, aspergillus flavus and rhizopus oryzae, and screened and verified by repeated tests, and have high hybridization specificity and accuracy. The chip provided by the invention ensures simple detection operations and relatively lower cost, saves time, is applied to the screening of the clinical common pathogenic microorganisms, the early rapid diagnosis of critical patients and the diagnosis of suddenly occurring infection event pathogens and favorable for clinically rapidly diagnosing infection sources and functions in the early detection and early diagnosis of clinical diseases.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a gene chip for detecting common clinical pathogenic microorganisms. Background technique [0002] Infectious disease has always been one of the most common clinical pathogenic factors, and it is also a problem that has plagued the diagnosis and treatment of clinical diseases for a long time. In addition to the increasing number of patients infected with various viruses such as HIV and hepatitis B, the most common clinical infection is still caused by various common bacteria and fungi. With the emergence of global aging, population mobility increases, and the prevalence and spread of pathogenic microorganisms increase, causing infectious diseases to present a trend of multiple and multiple infections. Due to the mutation of the pathogen itself and the resurgence of pathogens that have disappeared or been controlled, clinical infectious diseases are becoming more and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
CPCY02A50/30
Inventor 邓冠华郑璇周新孙靖谢焱胡一敏刘松梅郑芳涂建成袁媛丁峰王春芳
Owner GUANGZHOU IMPROVE MEDICAL TECH CO LTD
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