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Detection kit for Neosporiasis

A technology for neosporosis and a kit, which is applied in the field of molecular biology detection, can solve the problems of unsuitable detection, cumbersome operation, and lengthy process.

Inactive Publication Date: 2016-01-06
XINJIANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Traditional immunohistochemical techniques, ultrastructural detection techniques, and pathogen isolation techniques are lengthy and cumbersome, and are not suitable for the detection of this disease.

Method used

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  • Detection kit for Neosporiasis
  • Detection kit for Neosporiasis
  • Detection kit for Neosporiasis

Examples

Experimental program
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Effect test

example 1

[0026] Example 1. Design of neosporosis detection primers and fluorescent probes

[0027] ① Refer to the literature to understand the relevant characteristics of the Neospora NcSRS2 gene, download the Neospora NcSRS2 gene sequences uploaded in GenBank from different countries and regions, use DNAMAN software to compare and analyze all the sequences, and find out the conserved sequence of the NcSRS2 gene. The detection primers and fluorescent probes were designed within the sequence, and the design principles followed the Real-TimePCR primer amplification product length of 80-150bp. ② Use PrimerExpress3.0 and Oligo6.24 software to design detection primers NcSRS2F / R and MGB fluorescent probe NcSRS2P according to the sequence of the conserved region of Neospora NcSRS2 gene (accession number: U93870.1), and the 5' end of the probe is labeled with FAM fluorescent reporter Group, 3' labeled MGB fluorescence quencher group, amplified 146bp target fragment. The upstream primer NcSRS2...

example 2

[0028] Example 2: Preparation of target gene fragment positive plasmid

[0029] ① First, the designed detection primers were entrusted to Beijing Dingguo Changsheng Biotechnology Co., Ltd. for synthesis, and the synthesized primers were used for routine PCR amplification of Neospora positive DNA. The conventional PCR reaction system was 25 μL: 10×PCRBuffer (Mg 2+ plus) 2.5 μL, dNTP (2.5mM) 2 μL, upstream and downstream primers (10pM) 1 μL each, TaqDNA polymerase (5U / μL) 0.15 μL, template DNA 1.5 μL, ddH 2 O to make up 25 μL. The cycle conditions were: pre-denaturation at 94°C for 5min; denaturation at 94°C for 30s, annealing at 56°C for 30s, extension at 72°C for 30s, 35 cycles; extension at 72°C for 5min. ② Take 1 μL of 6×DNALoadingBuffer and add it to 5 μL of PCR product and mix well, and analyze the size of the amplified band by electrophoresis on 1.5% agarose gel and 100V for 40 minutes. If the band size is 146bp, follow-up experiments can be continued (see attached fig...

example 3 3D

[0030] Example 3. 3D digital PCR screening for optimal annealing-extension temperature and absolute quantitative analysis of amplified templates

[0031] ① Using NcSRS2F / R and NcSRS2P to extract 10 positive plasmids (32.5ng / μL) -6 、10 -7 、10 -8 Dilute the titer for 3D digital PCR amplification to screen the optimal annealing-extension temperature of primers and probes and determine the concentration of templates involved in the reaction. ② 3D digital PCR reaction system is 15 μL: 2×3DMasterMix 7.5 μL, primers, probes (10pM) 1 μL each, plasmid template 2 μL, ddH 2 O to make up 15 μL. The optimal cycle conditions for screening were: pre-denaturation at 96°C for 10 minutes; annealing-extension at 58°C for 2 minutes, denaturation at 98°C for 30 seconds, 40 cycles; extension at 60°C for 2 minutes. Therefore, 58°C is used as the optimal annealing-extension temperature for Real-TimePCR amplification (see attached figure 2 ); yields 10 -6 In the diluted plasmid template, the te...

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Abstract

The invention belongs to the technical field of molecular biological detection and relates to a (Real-Time PCR) detection kit for Neosporiasis. The detection kit is established based on 3D digital PCR scalar positive control and is applied to the clinical diagnosis and the daily monitoring of animals infected with the Neosporiasis. The detection kit comprises Neospora specific primers and a probe, wherein the Neospora specific primers are used for detecting; nucleotide sequences of the probe are represented by SEQ ID NO.1-3; a detected Neospora NcSRS2 gene is represented by SEQ ID NO.4. The detection kit has the characteristics of accurate detection, high sensitivity, strong specificity, simplty, convenience and rapidity, and is used for detecting DNA of polypides in an early infection stage and a recessive 'Neospora attached' period, thereby providing scientific technical supports for the clinical diagnosis and the daily monitoring of the Neosporiasis.

Description

field of invention [0001] The invention belongs to the technical field of molecular biology detection, and relates to a neosporidiosis (Real-TimePCR) detection kit based on a 3D digital PCR scalar positive control, which is used for clinical diagnosis and daily monitoring of neospora infection in different animals. monitor. Background technique [0002] Neosporiasis (Neosporiasis) is a newly discovered protozoan disease in 1998. The pathogen is Neospora caninum (Neosporacaninum), which parasitizes in the nucleated cells of various mammals, and can cause abortion, weak birth, stillbirth, etc. in pregnant dams, and movement disorders and nervous system diseases in newborn animals. Especially sensitive. Dogs are the terminal host and intermediate host of Neospora. Cattle, horses, sheep, deer and other domestic animals and various wild animals can be used as intermediate hosts. Healthy animals swallow oocysts or tachyzoites sporulated by Neospora. Horizontal infection occurs ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/90
CPCC12Q1/6893C12Q1/6851C12Q2531/113C12Q2545/113C12Q2561/101C12Q2561/113
Inventor 巴音查汗·盖力克陈千林王振宝郭庆勇张扬刘梦丽许正茂史亚明宋瑞其明·乌尔娜
Owner XINJIANG AGRI UNIV
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