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Internal amplification control (IAC) multiple polymerase chain reaction (PCR) detection kit for bovine neosporosis and toxoplasmosis

A technology of bovine neosporosis and detection kit, which is applied in the field of animal inspection and quarantine, bovine neosporosis and toxoplasmosis internal standard multiplex PCR detection kit

Inactive Publication Date: 2013-11-27
新疆出入境检验检疫局检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] After searching, there is no internal standard multiplex PCR detection with high sensitivity, good specificity, convenient operation, and the addition of internal standard to indicate and calibrate false negative results at home and abroad, which can simultaneously detect bovine neosporosis and toxoplasmosis method reporting

Method used

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  • Internal amplification control (IAC) multiple polymerase chain reaction (PCR) detection kit for bovine neosporosis and toxoplasmosis
  • Internal amplification control (IAC) multiple polymerase chain reaction (PCR) detection kit for bovine neosporosis and toxoplasmosis
  • Internal amplification control (IAC) multiple polymerase chain reaction (PCR) detection kit for bovine neosporosis and toxoplasmosis

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Experimental program
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Embodiment 1

[0063] Embodiment 1: Preparation of internal standard multiplex PCR detection kit for bovine neosporosis and toxoplasmosis

[0064] An internal standard multiplex PCR detection kit for bovine neosporosis and toxoplasmosis. The internal standard multiplex PCR detection kit is obtained through the following specific methods.

[0065] (1) Design and synthesis of primers

[0066] According to the Neospora Nc-5 gene with GenBank accession number X84238.1, the Toxoplasma gondii GRA6 gene with GenBank accession number HM776942.1 and the bovine prolactin PRL gene with GenBank accession number AF426315.1, a pair of specific Primers, the lengths of the amplified target fragments are 222 bp, 124 bp and 308 bp respectively.

[0067] (2) Preparation of template DNA and construction of positive recombinant plasmids

[0068] Nucleic acid was extracted according to the anticoagulant whole blood genome DNA extraction system. The liver, kidney, aborted fetus and other tissues were ground an...

Embodiment 2

[0091] Example 2: Preparation of Template and Construction of Positive Recombinant Plasmid

[0092] 1. Design and synthesis of primers and probes

[0093] The code numbers, sequences and positions of the primers of the present invention are shown in Table 1. Among them, refer to the Neospora Nc-5 gene (GenBank accession number: X84238.1), Toxoplasma gondii GRA6 gene (GenBank accession number: HM776942.1) and bovine Primers were designed for the prolactin PRL gene (GenBank accession number: AF426315.1), and the lengths of the amplified target fragments were 222 bp, 124 bp, and 308 bp. public channels.

[0094] Table 1 Design results of internal standard multiplex PCR primers

[0095]

[0096] 2. Preparation of template DNA and construction of positive recombinant plasmids

[0097] 2.1 Preparation of template DNA

[0098] Anticoagulated whole blood was operated according to the instructions of the blood genome DNA extraction system to extract nucleic acid. The liver, ki...

Embodiment 3

[0109] Embodiment three: the establishment of internal standard multiplex PCR amplification system

[0110] 1. Optimization of singleplex PCR reactions

[0111] A 20 μL reaction system was used: 10×buffer 2.0 uL, dNTP concentrations were 2.5, 2.0, 1.5 and 1.0 mmol / L, primer concentrations were 0.75, 0.5, 0.25 and 0.125 μmol / L, Mg 2+ The concentrations were 2, 1.5, 1.0 and 0.5 mmol / L, the Taq DNA polymerases were 1.75, 1.5, 1.25, 1.0, 0.75 and 0.5 U, and the annealing temperatures were 55 °C, 56 °C, 57 °C, 58 °C, 59 °C ℃ and 60 ℃, and the cycle parameters were 30, 35 and 40, respectively. When one item is optimized, the parameters of other items remain unchanged. Optimized single-plex PCR reaction conditions: 10×buffer 2.0 uL, dNTP concentration 1.5 mmol / L, Nc-5, GRA6 and PRL primer concentration 0.5 μmol / L, Mg 2+ The concentration is 0.5 mmol / L, Taq Taq DNA polymerase 1.0 U, DNA 2 μL, add deionized water to 20 μL system; amplification conditions: 95 ℃, 5 min; 95 ℃, 3...

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Abstract

The invention discloses an internal amplification control (IAC) multiple polymerase chain reaction (PCR) detection kit for bovine neosporosis and toxoplasmosis. An IAC which indicates false negative is introduced into a double PCR system; primer design and PCR amplification are performed, and reaction conditions are optimized; the reaction conditions are that dNTP concentration is 2.0 mmol / L, Mg<2+> concentration is 1.0 mmol / L, the concentrations of upstream and downstream primers corresponding to three genes Nc-5, GRA6 and PRL are 0.125 mmol / L, 0.5 mmol / L and 0.25 mmol / L respectively, and Taq DNA polymerase is 1.5 U; the amplification is performed at 95 DEG C for 5 minutes, at 95 DEG C for 30 seconds, at 58 DEG C for 45 seconds and at 72 DEG C for 30 seconds, and 30 circulations is carried out; finally extension is performed at 72 DEG C for 10 minutes. The kit is applied to the field of synchronous detection of bovine neosporosis and toxoplasmosis.

Description

[0001] technical field [0002] The invention relates to the technical field of animal inspection and quarantine. Specifically, the invention relates to the technical field of an internal standard multiple PCR detection kit for bovine neosporidiosis and toxoplasmosis. Background technique [0003] Bovine Neosporosis ( Neosporosis ) is caused by Neospora caninum ( Neospora caninum , N. caninum ) is a protozoan disease caused by parasitizing in the host body. The intermediate hosts of the disease are warm-blooded animals such as cattle, sheep, goats, dogs, horses, deer and mice, and the ultimate host is dogs. It mainly causes abortion, stillbirth and The disease of the motor nervous system of newborn animals can be transmitted vertically and occurs all year round. The disease is particularly harmful to cattle and is one of the main reasons for abortion, weak fetuses, stillbirths, and mummified fetuses in many areas of the world. bovine toxoplasmosis ( Toxoplasmosis ) i...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 季新成彭武丽王振宝于学辉史茜王科珂员丽娟段晓东
Owner 新疆出入境检验检疫局检验检疫技术中心