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Gene chip and kit for detecting common pathogen in dairy products

A gene chip and kit technology, which is applied in the field of gene chips and kits for detecting common pathogenic bacteria in dairy products, can solve problems such as difficult identification, and achieve the effects of simple operation, strong repeatability and high accuracy

Active Publication Date: 2008-08-13
TIANJIN BIOCHIP TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bacterial microorganisms can be identified to 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 and kit for detecting common pathogen in dairy products
  • Gene chip and kit for detecting common pathogen in dairy products
  • Gene chip and kit for detecting common pathogen in dairy products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Design and preparation of probe

[0034] 1. Sequence acquisition:

[0035] (1) Acquisition of bacterial 16s rDNA sequence: Download all 16s rDNA sequences of ten bacteria from the GenBank public database.

[0036] (2) Acquisition of 16S-23S rDNA interregion sequence: Download from the GenBank public database to obtain all 16S-23S rDNA interregion sequences of Salmonella, Staphylococcus aureus, Streptococcus pyogenes, Enterobacter sakazakii and their relatives .

[0037] The public database of Klebsiella pneumoniae's interregion sequence has only 21 interregion sequences of 9 strains, and the interregion sequence of Klebsiella oxytoca has only 1 sequence of one strain, which is not sufficient for screening specific probes. Need for needles. Our laboratory has tested 22 strains of Klebsiella pneumoniae, 3 strains of Klebsiella odorifera, 2 strains of Klebsiella rhinosclerosus, 4 strains of Klebsiella oxytoca, and 1 strain of Klebsiella terrestris. Bacteria and 1 str...

Embodiment 2

[0052] Example 2 Design and preparation of primers

[0053] 1. Sequence acquisition: same as the sequence of the designed probe.

[0054] 2. Design primers:

[0055] (1) Design of primers for the sequence of the amplified interregion: 16S rDNA of 10 bacteria downloaded from the public database NCBI is aligned with the sequence alignment software Glustal X, and a 16s rDNA conservative sequence near the interregion is selected as the upstream primer , The length conforms to the Tm value of 50℃±5℃, the length of 17bp±2bp, Hairpin: NONE, Dimer: NONE, False Priming: NONE, CrossDimer: NONE, and includes the bacterial universal probe sequence.

[0056] (2) Design of primers for amplifying ipaH gene sequence: align the ipaH gene sequences of the four Shigella species downloaded from the GenBank public database with the sequence alignment software Glustal X to find the conservative segment of the gene, Import the conservative segment into the primer design software PrimerPremier 5.0, and...

Embodiment 3

[0065]Example 3 Gene chip preparation-chip spotting

[0066] 1. Dissolving the probes: the probes synthesized in Example 1 were respectively dissolved in 50% DMSO solution, and diluted to make the final concentration of the probe reach 1 μg / μl.

[0067] 2. Add plate: add the dissolved probe to the corresponding position of the 384-well plate, 10μl per well.

[0068] 3. Spotting: Place a clean aldehyde-based glass slide (CEL Associates, Inc.) of 57.5mm×25.5mm×1mm (length×width×height) as shown in Figure 1 on the chip spotter (Spotarray 72). ), use SpotArray's control software (Tele chem smp3 stealty pin), run the program, and place dots on the aldehyde-based glass slide in a 4.5mm×4.5mm spot area according to the arrangement shown in Figure 2. , Forming a medium and low density DNA micro-matrix, the six dot matrix areas on the glass slide are arranged in the same pattern. The size of the dot matrix area is 3mm×2.25mm, the dot pitch in the dot matrix is ​​250μm, the matrix: 12×9, 1...

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Abstract

The present invention provides a gene chip for detecting common pathogen in dairy, which comprises a solid-phase vector and an oligonucleotide probe fixed on the solid-phase vector, wherein the oligonucleotide probe includes DNA fragment or its complementary DNA or RNA sequence selected from E. sakazakii, streptococcus pyogenes, staphylococcus aureus, Klebsiella pneumoniae, Klebsiella oxytoca, Listeria monocytogenes, salmonella and 16S-23S rDNA intergenic spacer region of Bacillus cereus, as well as ipaH gene of Shigella or gyrB gene of citrobacter freundii. The invention further provides a reagent box which uses the gene chip to detect pathogen in dairy. The gene chip and the reagent box of the invention for detecting pathogen in dairy are easy to operate, highly precise and greatly repeatable.

Description

Technical field [0001] The invention relates to a gene chip and a detection kit, in particular to a gene chip and a kit for detecting common pathogenic bacteria in dairy products Background technique [0002] Since the "inferior milk powder" incident occurred in Fuyang, Anhui in April 2004, the safety of milk powder has become the focus of widespread concern from all walks of life. The main consumer groups of milk powder are infants, children, the elderly or patients with low immunity, especially infants and young children are important consumer groups of milk powder. Therefore, the safety of milk powder is particularly important and has been affected by the State Council, functional departments and all levels. The government attaches great importance. In 2002, the Ministry of Science and Technology issued the "Tenth Five-Year Plan" National Major Scientific and Technological Dairy Special Project, which conducted rapid detection methods for several common pathogens in dairy prod...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12M1/34
CPCY02A50/30
Inventor 曹勃阳高旗利王敏王磊
Owner TIANJIN BIOCHIP TECH CO LTD
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