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Primer sequence for detecting Brucella based on dual priming oligonucleotide (DPO) primer, and detection kit thereof

A DPO primer, the technology of Brucella, applied in the direction of microorganism-based method, microbial determination/inspection, introduction of foreign genetic material using vectors, etc., can solve the problems of time-consuming and laborious, and achieve the effect of good repeatability and stability

Inactive Publication Date: 2013-07-24
哈尔滨海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional PCR primer design not only needs to repeatedly compare the specificity of the primers, but also needs to optimize the parameters and reaction conditions of the primers, especially the annealing temperature, to prevent non-specific amplification, especially when multiplex PCR is involved. Experiments to verify the specificity of the detection method, time-consuming and laborious

Method used

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  • Primer sequence for detecting Brucella based on dual priming oligonucleotide (DPO) primer, and detection kit thereof
  • Primer sequence for detecting Brucella based on dual priming oligonucleotide (DPO) primer, and detection kit thereof
  • Primer sequence for detecting Brucella based on dual priming oligonucleotide (DPO) primer, and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Preparation of Brucella DNA template

[0045] Extract and purify bacterial genomic DNA according to the instructions of TIANamp Bacteria DNA Kit, and add the corresponding reagents in order:

[0046] (1) Take 1.5 mL of bacterial liquid, centrifuge at 10,000 r / min for 1 min, discard the supernatant, add 200 μL of buffer GA, and suspend the bacterial cells thoroughly;

[0047] (2) Add 20 μL proteinase K (20mg / mL), and add 220 μL buffer solution GB, mix thoroughly, and act in a 70°C water bath for 10 minutes;

[0048] (3) Add 220 μL of absolute ethanol, mix thoroughly for 15 seconds, transfer the resulting solution (including flocculent precipitate) to the adsorption column CB3 after brief centrifugation, centrifuge at 12000 r / min for 30 seconds, and discard the liquid in the collection tube;

[0049] (4) Add 500 μL buffer GD (containing absolute ethanol) to the adsorption column CB3, centrifuge at 12000r / min for 30s, and discard the liquid in the collection tube;

[005...

Embodiment 2

[0054] Example 2 Design and synthesis of PCR primers

[0055] Select Brucella ( Brucella ) Omp25 gene was used as the target gene, and the following primers were designed and synthesized to amplify the target gene:

[0056] (1) Preparation of PCR amplification primers for the positive control of Brucella Omp25 gene:

[0057] BO-F: 5'-TCGTAATCGTCTCGGCTGCGT-3',

[0058] BO-R: 5'-GGATGTTGTCCGTCAGCTTGG-3'.

[0059] (2) For Brucella ( Brucella ) Omp25 gene sequence for BLAST analysis, select the conserved region of the target gene sequence to design and synthesize DPO identification primers (PCR product size is 405bp), the nucleotide sequence of the primers is:

[0060] BO-DPOF: 5′-CTTTTGCTGCCGACGCCATCCIIIIIIAGGAACAGC-3′,

[0061] BO-DPOF: 5'-TTCGTCGTCCAAGCCGTTGTTAAIIIIIGCTTGATCT-3'.

Embodiment 3

[0062] Example 3 Preparation of positive control substance

[0063] 10×PCR Buffer 2.5μL dNTP (2.5mM for each) 2.0 μL Upstream primer BO-F (10μM) 1.0 μL Downstream primer BO-R (10μM) 1.0 μL Taq DNA Polymerase (5U / μL) 0.5μL DNA template 0.5μL wxya 2 o 17.5μL

[0064] The reaction conditions of PCR were: 95°C for 5min; 35 cycles of 94°C for 30s, 60.5°C for 45s, and 72°C for 45s; 72°C for 10min.

[0065] The PCR amplification result was detected by agarose gel electrophoresis, and the size of the PCR product was 501bp.

[0066] Connect the PCR product to the pMD-19-T vector, transform competent Escherichia coli JM109, and obtain positive recombinant bacteria, and prepare positive plasmids according to the instructions of the Huashun Mini-Plasmid DNA Extraction Kit:

[0067] (1) Pick a single colony of positive clones and inoculate in 5 mL containing 100 μg / mL Amp r In LB medium, cultivate overnight at 37°C;

[0068]...

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Abstract

The invention discloses a polymerase chain reaction (PCR) method for detecting Brucella based on a dual priming oligonucleotide (DPO) primer, and a DPO primer sequence thereof. The method comprises the following steps of: selecting a Brucella Omp25 gene as a target gene; through BLAST analysis of the gene sequence, selecting a conserved region of the target gene sequence, and designing and synthesizing the DPO primer, wherein the nucleotide sequence of the DPO primer is as follows: BO-DPOF: 5'-CTTTTGCTGCCGACGCCATCCIIIIIAGGAACAGC-3', and BO-DPOR: 5'-TTCGTCGTCCAAGCCGTTGTTAAIIIIIGCTTGATCT-3'; and establishing a DPO-PCR detection method so as to perform precise qualitative detection on the Brucella. The invention also relates to a detection kit which has the advantages of high detection specificity, high accuracy and high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a primer sequence for detecting Brucella based on DPO primers and a detection kit thereof. Background technique [0002] Brucellosis ( Brucella ) is a type of Gram-negative short bacillus that can infect a variety of domestic and wild animals, mainly causing infectious abortion in female animals. It can also infect people at the same time, especially Brucella sheepis is the greatest threat to people, causing similar clinical symptoms and pathological damage, such as orchitis or epididymitis, infertility, inflammation of reproductive organs and fetal membranes, miscarriage, infertility, joint Inflammation, bronchitis and local lesions of various tissues, etc., lead to huge economic losses and serious public health problems. [0003] Existing detection methods for Brucella: For bacteriological testing, during initial isolation, it must grow in an envir...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12N15/63C12N15/66C12R1/01
Inventor 徐义刚李丹丹张柏棋刘忠梅李苏龙
Owner 哈尔滨海关技术中心
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