PCR (Polymerase Chain Reaction) kit and primers for detecting swine dysentery

A swine dysentery and reagent kit technology, applied in the field of inspection and quarantine, can solve the problems that the laboratory is difficult to meet the detection requirements, there is no satisfactory detection method, and the detection conditions are high, and it is beneficial to diagnosis and monitoring, easy to popularize and apply, and easy to master and the effect of the operation

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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  • PCR (Polymerase Chain Reaction) kit and primers for detecting swine dysentery
  • PCR (Polymerase Chain Reaction) kit and primers for detecting swine dysentery
  • PCR (Polymerase Chain Reaction) kit and primers for detecting swine dysentery

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Design and synthesis of primers

[0068] According to the relevant gene sequences of T. hyodysenteriae registered in Genbank, the DNAMAN software was used for comparison and analysis, and the highly conserved regions of the tlyA gene were selected, and the primers for T. hyodysenteriae were designed with Oligo6.0 software. Same as Table 1.

[0069] Primers were synthesized by Shanghai Xuguan Biotechnology Co., Ltd.

Embodiment 2

[0070] Example 2: Extraction of Brevi / Treponema hyodysenteriae DNA

[0071] The DNA of Brevi / Treponema hyodysenteriae was extracted with DNA extraction reagent.

[0072] The specific operation is as follows:

[0073] 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.

[0074] 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 respectively, and use a tip for each sample; shake and mix for 5 seconds on the mixer. Centrifuge at 13000 r / min for 10 min at 4°C~25°C.

[0075] 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.

[0076] ④ Dry bath or boiling water bath at 100 ℃ for...

Embodiment 3

[0078] Embodiment 3: The establishment of PCR system

[0079] 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 PCR was established.

[0080] According to the method of Example 2, the Brevi / Treponema hyodysenteriae DNA was obtained, and the template concentration was about 10 4 -10 6 copies / microliter, perform PCR, and optimize the reaction system.

[0081] (1) Optimization of primer concentration

[0082] Probes and primers were optimized separately. The primer concentrations from 0.1 μM to 0.8 μM in increments of 0.1 μM and the probe concentrations from 0.1 μM to 0.5 μM in increments of 0.1 μM were cross-matched for real-time PCR to select the optimal primer and probe concentrations. The reaction system conditions are shown in Table 2, and the primers and probe sequences used are shown in Table 1:

[0083] Table 2 Primer probe optimization PCR reactio...

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) kit and primers for detecting swine dysentery. A group of primers for detecting the swine dysentery have the nucleotide sequences disclosed in a sequence table SEQIDNo: 1 and a sequence table SEQIDNo: 2. The invention has the advantages that compared with etiology separation culture, the PCR technology applied in the detection of the swinedysentery is faster and more convenient, the bacterium separation culture method at least needs 10 days and hard culture conditions, strict anaerobic culture is needed, and the PCR can be completed only in 5 hours; and compared with etiology separation, the PCR needs relatively simple equipment, is more convenient to master and operate from a technical perspective, is more convenient to apply andpopularize in areas with insufficient conditions and is more conductive to the diagnosis and monitoring of the swine dysentery.

Description

technical field [0001] The invention relates to a PCR kit and primers for detecting swine dysentery, 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 that of normal pigs, and the weight gain ...

Claims

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Application Information

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