Detection gene chip and detection method of main pathogenic bacteria of dairy cow mastitis

A technology for detecting gene chips and cow mastitis, applied in the field of detection, can solve problems such as unseen technical reports and solutions, difficult practical operations, inability to accurately detect pathogenic bacteria, etc., to achieve easy promotion and use, and low false positive rate , the effect of high accuracy

Inactive Publication Date: 2012-02-22
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Chinese patent application 200710166530.3 discusses the detection gene chip of pathogenic bacteria in milk products, fluorescently labeled on the probe, the probe is obtained by screening between 16S-23S rDNA, but the interval is on

Method used

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  • Detection gene chip and detection method of main pathogenic bacteria of dairy cow mastitis
  • Detection gene chip and detection method of main pathogenic bacteria of dairy cow mastitis
  • Detection gene chip and detection method of main pathogenic bacteria of dairy cow mastitis

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 The rapid detection experiment of the main pathogenic bacteria of dairy cow mastitis

[0043] 1. The instruments and reagents are:

[0044] iCycler PCR instrument (Bio-Rad company), GenePix 4000B scanner (Axon company), PixSys 5000 spotting instrument (Cartesian company), 8909 DNA synthesizer (ABI company), DU640 ultraviolet spectrophotometer (Beckman company), Gel Doc1000 gel imager (Bio-Rad). DNA synthesis reagents were purchased from Glen Research Company in the United States, PCR-related reagents were purchased from Shanghai Bioengineering Co., Ltd., gene chip substrates were purchased from CEL Associates Company, and Cy3 fluorescent dye was purchased from Amersham Company. Rabbit blood agar medium, LB liquid medium, LB solid medium, and other reagents are all products of Promega.

[0045] 2. Experimental steps

[0046] (1) Prepare the DNA of the sample strain and the standard strain;

[0047] Preparation of DNA of standard strains of 6 main pathogenic...

Embodiment 2

[0080] Embodiment 2 evaluation experiment

[0081] In order to evaluate the ability of the present invention's method to clinical sample detection, respectively collect and separate 78 clinical samples from Guangdong Zengcheng Dairy Farm and Guangzhou Huamei Dairy Farm, take 8ul overnight culture supernatant as template and carry out PCR amplification and gene chip Hybridization, and compared with conventional methods of isolation, purification, culture, and genus identification. The results are shown in Table 3. Table 3 shows that compared with the conventional culture method, the detection results of the conventional method of 9 samples were inconsistent with the gene chip method, 1 case was Streptococcus agalactiae, 5 cases were Escherichia coli; the other 3 cases were strange deformation Bacillus, the coincidence rate of the gene chip detection established in the experiment reached 89.46%.

[0082] Table 3 Comparison results of gene chips and conventional detection method...

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Abstract

The invention provides a detection gene chip and a detection method for the main pathogenic bacteria of cow mastitis. The gene chip includes a solid phase carrier and a specific detection probe fixed on the solid phase carrier, and the specific detection probe includes 6 kinds of universal primer sequences designed with the Escherichia coli 16S rDNA conserved region as the base sequence. A specific oligonucleotide hybridization probe designed in the variable region of the main pathogenic bacteria of dairy cow mastitis; the six main pathogenic bacteria of dairy cow mastitis are Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, pneumoniae Klebsiella, Proteus mirabilis, and Staphylococcus aureus. The invention precisely limits the annealing temperature of the PCR reaction, optimizes the hybridization conditions of the PCR-amplified nucleic acid fragment and the gene chip, obtains a good hybridization effect, and finally obtains accurate detection results. The invention has good specificity, high accuracy rate, reliable stability, low false positive rate of detection results, simple operation steps and easy popularization and use.

Description

technical field [0001] The invention belongs to the technical field of detection and relates to a rapid detection technology, in particular to a detection gene chip and a detection method for the main pathogenic bacteria of cow mastitis. Background technique [0002] Cow mastitis not only seriously affects the quality of milk, causes the productivity of dairy cows to drop seriously and even be eliminated, but also endangers human health. The epidemic of dairy cow mastitis has become a recognized public health, social and economic problem. There are many types of pathogenic bacteria that cause mastitis in dairy cows. At present, there are more than 220 known pathogenic bacteria, of which more than 20 are common, and 90% of the pathogenic bacteria of cow mastitis are mainly Escherichia coli, Staphylococcus aureus Coccus, Staphylococcus epidermidis, Klebsiella pneumoniae, Proteus mirabilis, Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus uberis, Salmonella, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 李守军杨林邢会杰
Owner SOUTH CHINA AGRI UNIV
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