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Real-time fluorescence PCR (polymerase chain reaction) kit and oligonucleotide sequence for detecting swine dysentery

A nucleotide sequence, oligonucleotide technology, applied in fluorescence/phosphorescence, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of lack of satisfactory detection methods, high detection conditions, and difficult laboratory requirements. Detection requirements and other issues to achieve the effect of improving specificity, reducing workload and improving work efficiency

Inactive Publication Date: 2012-03-14
PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing detection conditions have high requirements (strict anaerobic culture), and it is difficult for general laboratories to meet the detection requirements
[0006] So far, there is no satisfactory simple and feasible detection method for the disease

Method used

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  • Real-time fluorescence PCR (polymerase chain reaction) kit and oligonucleotide sequence for detecting swine dysentery
  • Real-time fluorescence PCR (polymerase chain reaction) kit and oligonucleotide sequence for detecting swine dysentery
  • Real-time fluorescence PCR (polymerase chain reaction) kit and oligonucleotide sequence for detecting swine dysentery

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Design and synthesis of Taqman probes and primers

[0050] According to the relevant gene sequences of Treponema hyodysenteriae registered in Genbank, the DNAMAN software was used for comparison and analysis, and the highly conserved regions of the nox gene were selected, and Primer Express V2.0 software was used to design probes for Treponema hyodysenteriae and primers. Same as Table 1.

[0051] Primers and probes were synthesized by Shanghai Xuguan Biotechnology Co., Ltd.

Embodiment 2

[0052] Example 2: Extraction of Brevi / Treponema hyodysenteriae DNA

[0053] The DNA of Brevi / Treponema hyodysenteriae was extracted with DNA extraction reagent.

[0054] The specific operation is as follows:

[0055] ① Take n 1.5 mL sterilized centrifuge tubes, where n is the sum of the number of samples to be tested, one tube of positive control and one tube of negative control, and number each tube.

[0056] ② Add 200 μL of DNA Extraction Solution I to each tube, then add 200 μL each of the sample to be tested, the negative control and the positive control, and use a tip for each sample; shake and mix for 5 s on a mixer. Centrifuge at 13000 r / min for 10 min at 4°C~25°C.

[0057] ③ Aspirate and discard the supernatant as much as possible without touching the precipitate, then add 10 μL of DNA Extraction Solution II, shake and mix for 5 s on a mixer. Centrifuge at 2000 r / min for 10 s at 4°C~25°C.

[0058] ④ Dry bath or boiling water bath at 100 ℃ for 10 minutes; centrifu...

Embodiment 3

[0060] Embodiment 3: the establishment of real-time fluorescent PCR

[0061] 1. The concentration of probes and primers, the concentration of magnesium ions and the concentration of enzyme were optimized respectively, and the reaction system of real-time fluorescent PCR was established.

[0062] According to the method of Example 2, the Brevi / Treponema hyodysenteriae DNA was obtained, and the template concentration was about 10 4 -10 6 copy / microliter, perform real-time fluorescent PCR, and optimize the reaction system.

[0063] (1) Optimization of probe and primer concentrations

[0064] Probes and primers were optimized separately. The primer concentration increased by 0.1 μM interval from 0.1 μM to 0.8 μM and the probe concentration increased by 0.1 μM interval from 0.1 μM to 0.5 μM were cross-matched for real-time fluorescent PCR to select the optimal primer and probe concentration. The reaction system conditions are shown in Table 2, and the primers and probe seque...

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Abstract

The invention discloses a real-time fluorescence PCR (polymerase chain reaction) kit and an oligonucleotide sequence for detecting swine dysentery. The oligonucleotide sequence for detecting swine dysentery is an oligonucleotide sequence shown from SEQ ID No.1 to SEQ ID No.3 in a sequence table. The invention also provides a detection kit containing the oligonucleotide sequence. The kit and the oligonucleotide sequence have the advantages that: when the real-time PCR technology is applied to swine dysentery detection, the detection specificity and sensitivity are further improved, the workload is reduced, the working efficiency is improved, and a target fragment can be quantitatively detected. By virtue of optimization of reaction conditions, the real-time PCR can detect pathogen amount of 10<2> copy / reaction minimally. 318 clinical samples from 7 pig farms are detected by using the method, and experiment results prove that: the method is a specific, sensitive and efficient detection method. The technology has great application prospect in import and export sample quarantine, as well as monitor and diagnosis of clinical plague.

Description

technical field [0001] The invention relates to a kit for detecting swine dysentery by real-time fluorescent PCR and its primers and probes, belonging to the field of inspection and quarantine. Background technique [0002] Swine dysentery ;SD ) is a Gram-negative anaerobic spirochete, successively named Treponema hyodysenteriae ( Treponema hyodysenteriae ;T.h), Spirulina hyodysenteriae ( Serpulinahyodysenteriae; S.h). It is now collectively named Brachyspira hyodysenteriae ( Brachyspira hyodysenteriae ; B.h), the name Treponema hyodysenteriae is still widely used for historical reasons. The disease is an intestinal infection of pigs characterized by mucus or mucohemorrhagic diarrhea. The morbidity in pig herds is quite high, and the growth and development of sick pigs are hindered, and the consumption of feed increases, causing great economic losses to the pig industry in various places. According to statistics, the feed consumption rate of diseased pigs is twice t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N21/64
Inventor 张伟高志强李宁安健凌凤俊张鹤晓张利峰汪琳柏亚铎谷强乔彩霞蒲静吴丹赖平安
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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