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Gene chip for detecting pathogens of lower respiratory tract and reagent kit

A gene chip and pathogenic bacteria technology, applied in the field of gene chips and kits for detecting pathogenic bacteria of the lower respiratory tract, can solve problems such as identification difficulties, and achieve the effects of simple operation, strong repeatability and high accuracy

Active Publication Date: 2010-06-23
TIANJIN BIOCHIP TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bacterial microorganisms can be identified to a species or genus using 16S rRNA and 23S rRNA, but it is very difficult to identify bacterial species or genus with close relatives (BodrossyL, Sessitsch A. 2004. Oligonucleotide microarrays in microbial diagnostics. Current Opinion in Microbiology. 7:245-25)

Method used

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  • Gene chip for detecting pathogens of lower respiratory tract and reagent kit
  • Gene chip for detecting pathogens of lower respiratory tract and reagent kit
  • Gene chip for detecting pathogens of lower respiratory tract and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Design and preparation of embodiment 1 probe

[0033] 1. Sequence acquisition:

[0034] (1) Acquisition of bacterial 16s rDNA sequences: eight species of bacteria (Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Haemophilus influenzae, pneumoniae) were downloaded from the GenBank public database. Streptococcus, Legionella pneumophila and Moraxella catarrhalis) all 16s rDNA sequences.

[0035] (2) Acquisition of 16S-23S rDNA interregional sequences: download all 16S-23S rDNA interregional regions of Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii and their relatives from the GenBank public database sequence.

[0036]There are only 21 interval sequences of 9 strains in the public database of Klebsiella pneumoniae, and only 1 sequence of 1 strain of Klebsiella oxytoca, which cannot meet the requirements of screening specificity detection. needle needs. 22 strains of Klebsiella pneumoniae, 3 strains ...

Embodiment 2

[0051] Design and preparation of embodiment 2 primers

[0052] 1. Sequence acquisition: the same as the sequence of the designed probe.

[0053] 2. Design primers:

[0054] (1) Design of primers for amplifying the interregional sequence: After comparing the 16S rDNA of 8 species of bacteria downloaded from the public database NCBI with the sequence alignment software Glustal X, a 16S rDNA conserved sequence close to the interregional region was selected as the upstream primer , the length conforms to T m Value: 50°C±5°C, length: 17bp±2bp, Hairpin: NONE, Dimer: NONE, False Priming: NONE, Cross Dimer: NONE, and contains bacterial universal probe sequence.

[0055] (2) Design of primers for amplifying the copB gene sequence: compare the above-mentioned catamorra copB gene sequence downloaded from the GenBank public database with the sequence alignment software Glustal X to find the conserved segment of the gene, and then identify the conserved segment of the gene. The segmen...

Embodiment 3

[0062] Example 3 Gene Chip Preparation——Chip Spotting

[0063] 1. Dissolving probes: the probes synthesized in Example 1 were respectively dissolved in 50% DMSO solution, and diluted so that the final concentration of the probes reached 1 μg / μl.

[0064] 2. Adding plate: Add the dissolved probe to the corresponding position of the 384-well plate, 10 μl per well.

[0065] 3. Spotting: as figure 1The shown 57.5mm × 25.5mm × 1mm (length × width × height) clean aldehydated glass slide (CELAssociates, Inc.) was placed on the stage of the chip spotter (Spotarray 72), using the SpotArray Control software (Tele chem SMP3 stealth pin), run the program, press figure 2 The arrangement shown is spotted on the aldehydeized glass slide in the spotting area of ​​4.5 mm×4.5 mm to form a medium-low density DNA micro-array, and the array arrangement rules in the six dot matrix areas on the glass slide are the same. The size of the dot matrix area is 3mm×2.25mm, the dot pitch in the dot ma...

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Abstract

The invention provides a gene chip for detecting pathogens of lower respiratory tract, which comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, wherein the oligonucleotide probe comprises a sequence selected from one or more DNA sequences of staphylococcus aureus, 16s-23s rDNA intergenic spacer region of Klebsiella pneumoniae or Acinetobacter baumannii, 16S gene of pseudomonas aeruginosa or haemophilus influenzae, gyrB gene of streptococcus pneumoniae or legionella pneumophila and copB gene of moraxella catarrhalis or the complementary DNA or RNA sequence. The invention further provides a reagent kit containing the gene chip. The utilization of the gene chip and the reagent kit can detect the pathogens of the lower respiratory tract; furthermore, the operation is simple, the accuracy is high and the repeatability is strong.

Description

technical field [0001] The invention relates to a gene chip and a test kit for detection, in particular to a gene chip and a test kit for detecting lower respiratory tract pathogenic bacteria. Background technique [0002] Respiratory tract infection is one of the most common types of infectious diseases, accounting for about 30% to 40% of infectious diseases. Understanding the types of pathogenic bacteria that cause such diseases, especially cultivating and isolating pathogenic bacteria in time, is of great importance to the clinical diagnosis of the disease. Diagnosis, drug selection and prognosis are of great importance. [0003] Lower respiratory tract infection (tracheitis, bronchitis, bronchiolitis and pneumonia) is an infectious disease that seriously threatens the health of people in today's society. The danger is especially serious for the elderly and children who are immunocompromised. Epidemiological studies have shown that acute respiratory infection kills abou...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/04
CPCY02A50/30
Inventor 王磊姚英曹勃阳
Owner TIANJIN BIOCHIP TECH CO LTD
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