Gene chip for detecting common clinical pathogenic microorganism

A pathogenic microorganism and gene chip technology, which is applied in the field of gene chips for the detection of common clinical pathogenic microorganisms, can solve the problems of staying, unable to fully meet the needs of accurate detection of microorganisms, and a single probe, and achieves moderate length, reasonable probe design, high specific effect

Inactive Publication Date: 2012-10-10
WUHAN UNIV
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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 common clinical pathogenic microorganism
  • Gene chip for detecting common clinical pathogenic microorganism
  • Gene chip for detecting common clinical pathogenic microorganism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The preparation of embodiment 1 gene chip

[0063] This example uses a nylon membrane as a solid phase carrier to illustrate the preparation method of the gene chip of the present invention.

[0064] (1) With a positively charged nylon membrane as a carrier:

[0065] Prepare a positively charged nylon membrane in a size of 4 x 2 cm 2 Size, placed in a dry environment at room temperature, ready for use.

[0066] (2) Design primers and probes:

[0067] Firstly, the 16S rRNA gene and ITS gene sequences of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Candida albicans, Candida glabrata and Candida tropicalis were retrieved from the PUBMED online database by DNAMAN 5.2.2 (Lynnon Biosoft, USA) and NCBI BLAST software (http: / / www.ncbi.nlm.nih.gov / BLAST / ) are compared to find their conserved regions and hypervariable regions, and then apply Primer Premier 5.0 (PREMIER Biosoft International, CA) designed primers in these conserved regions and probes...

Embodiment 2

[0085] The detection of embodiment 2 pathogenic microorganisms

[0086] (1) Extract bacterial DNA

[0087] Staphylococcus aureus (ATCC25923), Klebsiella pneumoniae (ATCC700603), Pseudomonas aeruginosa (ATCC27853), Candida albicans (ATCC64548), and Candida glabrata (ATCC900030) were extracted by CTAB / NaCl method, respectively. and the pathogenic microorganism DNA of Candida tropicalis (CCTCC AY 92045) as templates.

[0088] (2) target DNA amplification

[0089] 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), each primer of 10 μmol / L, 10mmol / L dNTPs (Roche), 2U Taq DNA polymerase (Biostar), Biotin-11-dUTP (Fermentas) and sterile water composition. The PCR reaction conditions are: 50μl system (multiple PCR):

[0090] Taq buffer (10×) 5μl

[0091] dNTPs (10mM) 0.8μl

[0092] srr-rl 1.5 μl

[0093] srr-dg 1.5 μl

[0094] srr-ra 1.5 μl

[0095] srr-pg 1.5μl...

Embodiment 3

[0122] The detection of embodiment 3 pathogenic microorganisms

[0123] In this example, according to the method of Example 2, 6 strains were cultured and biochemically identified as Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Candida albicans, Candida tropicalis and Candida glabrata The pathogenic microorganisms were detected by clinical isolation (Department of Laboratory, Zhongnan Hospital, Wuhan University, Wuhan, Hubei). 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 a common clinical pathogenic microorganism, which comprises special detection probes fixed on a solid-phase vector. The probes are obtained through design for 16SrRNA genes and ITS gene sequences of staphylococcus aureus, klebsiella pneumoniae, pseudomonas aeruginosa, candida albicans, smooth candida krusei and candida tropicalis and through repeated selection by experiments and verification. The probes have high specific hybridization and accuracy. The invention also discloses the gene chip and a group of detection kit of universal primers. The gene chip of the invention is simple to operate in a whole detection process, saves time, has low cost, is suitable for the screening of the common clinical pathogenic microorganism, the early rapid diagnose for acute and severe patients and the diagnose for outbreak infection pathogen, contributes to clinical rapid diagnose for an infectious agent and plays a role in early detection and early diagnose.

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): C12Q1/68C12Q1/14C12Q1/04
Inventor 周新郑璇邓冠华谢焱胡一敏刘松梅郑芳涂建成
Owner WUHAN UNIV
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