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Method for detecting bovine neosporosis and application

A bovine neosporosis and detection method technology, which is applied in the detection of bovine neosporosis and protozoan parasitic diseases, achieving the effects of short detection time, guaranteed detection quality and high detection sensitivity

Inactive Publication Date: 2010-09-15
新疆出入境检验检疫局检验检疫技术中心
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] After searching, there is no internal standard co-amplification template and fluorescent probe constructed by bridging PCR amplification at home and abroad. The construction has high detection sensitivity, good specificity, convenient operation, and can indicate and calibrate false negative results, improving accuracy. The report of real-time quantitative PCR method for the detection of bovine neosporosis

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  • Method for detecting bovine neosporosis and application
  • Method for detecting bovine neosporosis and application
  • Method for detecting bovine neosporosis and application

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Experimental program
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Effect test

Embodiment 1

[0064] Embodiment one: the detection of bovine neosporosis

[0065] 1. Design and synthesis of primers and probes:

[0066] According to the N.caninumNc-5 gene sequence, two 138bp specific primers Np2 and Nr2, one NTpro fluorescent probe, and two template construction primers Np1 and Nr1 were amplified, and Np1 and Nr1 were located outside Np2 and Nr2. A fragment with a length of 338bp was obtained. The sequence of the probe NTpro was rearranged, and the primers Nc1 and Nc2 were designed by using the principle of base mutation, and the internal standard probe NCpro was constructed by bridging PCR amplification.

[0067] 2. Preparation of target DNA template:

[0068] Bovine anticoagulated whole blood and bovine aborted fetal tissue were extracted according to the Genomic DNA Extraction Kit and DNAzol from Biotec, respectively, and operated according to the actual instructions.

[0069] Using the whole genome DNA of Neospora caninum as template, using Np1 and Nr1 as amplific...

Embodiment 2

[0079] Embodiment 2: Obtaining the target template

[0080] Bovine anticoagulated whole blood and bovine aborted fetal tissue were extracted according to the Genomic DNA Extraction Kit and DNAzol from Biotec, respectively, and operated according to the actual instructions.

[0081] Select 25μL PCR reaction system: 2.5μL 10×Buffer, 1.5μL MgCl 2 (25mM), 2μL dNTP (2.5mM), 1μL each of upstream and downstream primers (10μM), 1μL Neospora caninum genomic DNA, 0.25μL Taq DNA polymerase (5U / μL), add ddH 2 O to 25 μL. The PCR reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 62°C for 20 s, extension at 72°C for 30 s, a total of 35 cycles; extension at 72°C for 10 min.

[0082] The amplified product was electrophoresed with 10g / L agarose, gel purified and recovered, connected to the pMD18-T vector, transformed into DH5α competent cells, and the plasmid was extracted for PCR, enzyme digestion and sequencing identification.

[0083] 1...

Embodiment 3

[0087] Embodiment three: the construction of internal standard template

[0088] The plasmid NT was used as a template, and primers Np1, Nc1, Nc2, and Nr1 were used as paired primers for PCR amplification. The amplification conditions were: 94°C for 5 minutes; 94°C for 30s, 62°C for 20s, 72°C for 30s, 35 cycles; 72°C ℃ 10min. The amplified products were named Np1c1 and Nc2r1, respectively.

[0089] Using plasmid NT as a template, primers Np1, Nc1 and Nc2, Nr1 were used as paired primers for PCR amplification, 25 μL reaction system, including 2.5 μL 10×PCR buffer, 1.5 μL MgCl 2 (25mM), 2μL dNTP (2.5mM) mixture, 0.25μL Taq DNA polymerase (5u / μL), 1μL each of upstream and downstream primers (10μM), 1μL template. The amplification conditions are: 94°C for 5 minutes; 35 cycles of 94°C for 30s, 62°C for 20s, and 72°C for 30s; 72°C for 10 minutes. Fragments of 194bp and 168bp were amplified respectively, and named Np1c1 and Nc2r1, see attached Figure 4 .

[0090] Np1c1 and Nc2r...

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Abstract

The invention discloses a method for detecting bovine neosporaosis. The method comprises the following steps of: adding an internal amplification control for indicating false negative into a PCR reaction system, constructing an interior label template by basic group rearrangement, primer design and bridging PCR amplification, constructing a fluorescent quantitative PCR reaction system containing internal label, and determining reaction conditions of a co-amplification system, wherein Mg2+ is 2.5mmol / L, the dNTP is 0.3mmol / L, the primer is 0.5mmol / L, and the probe is 0.2mmol / L; and the amplification conditions comprise 50 DEG C, 2min and one cycle, or 95 DEG C, 5min and one cycle, or 95 DEG C and 15s or 57 DEGC and 45s and 45 cycles. The method has the advantages of high detection sensitivity, good specificity and convenient operation, can indicate and correct the generation of false negative results, and is widely applied in the field of timely, accurately and effectively detecting the bovine neosporaosis.

Description

field of invention [0001] The invention belongs to the field of detection of animal epidemic diseases, in particular to a detection method of protozoan parasitic diseases, in particular to a detection method and application field of bovine neosporosis. Background technique [0002] Neosporosis is a protozoan disease caused by Neospora caninum parasitizing in the host. The disease can be transmitted vertically and is one of the main causes of abortion in dairy cows, which has caused great economic losses to animal husbandry. The disease is widely distributed worldwide. Since 2001, the presence of Neospora caninum antibodies has been detected in the serum of cows (yaks) in Beijing, Shanxi, Qinghai, Hebei, Xinjiang and other regions. So far, there are no effective drugs and vaccines to prevent and treat this disease. Therefore, good detection technology is a good way to effectively control this disease. [0003] Currently, the commonly used detection methods include indirect...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 季新成段晓东黄玲员丽娟窦辉牛国辉刘菲于学辉
Owner 新疆出入境检验检疫局检验检疫技术中心
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