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Specific detection primer for swine toxoplasmosis and detection kit

A technology for detection of toxoplasmosis and primers, applied in biochemical equipment and methods, measurement/testing of microorganisms, microorganisms, etc., can solve the problem of sensitivity, specificity and clinical application of unidentified pig toxoplasmosis infection Effect and other issues, to achieve high sensitivity, strong specificity, easy to determine the effect

Inactive Publication Date: 2017-06-13
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the currently reported B1 gene (Pelloux et al, 1998; Contini et al, 2002), the ribosomal DNA internal transcribed spacer (ITS) sequence (Jauregui et al, 2001; Xie Dehua et al, 2005) and the T. gondii genome Identified a 200-300 copies of a 529bp repetitive DNA fragment (Homan et al, 2000) as a genetic marker to establish a specific PCR diagnostic method for toxoplasmosis, but it has not been clearly determined in terms of sensitivity, specificity and clinical application in porcine toxoplasmosis Application Effect of Diagnosis and Infection of Entomosis

Method used

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  • Specific detection primer for swine toxoplasmosis and detection kit
  • Specific detection primer for swine toxoplasmosis and detection kit
  • Specific detection primer for swine toxoplasmosis and detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Example 1 Primer design and screening

[0051] The dense granule protein 14 (GRA14) gene was used as the target gene for the diagnosis of toxoplasmosis, and specific primers were designed according to the GRA14 gene sequence, as shown in Table 1.

[0052]

[0053] The PCR reaction system is: Premix E×Taq Version 2.0 (Loading dye Mix) 12.5 μL, forward primer and reverse primer 0.5 μL each, double distilled water 9.5 μL, template DNA 2 μL, number of amplified samples n (n=number of samples +3). Mix the above reaction solution in a centrifuge tube, mix well, and dispense. Take each sample DNA into the corresponding reaction tube, mark each reaction tube, mix and centrifuge, and place it on the PCR machine for reaction.

[0054] PCR amplification conditions are: (i) pre-denaturation at 94°C for 5 min; (ii) denaturation at 94°C for 30 s, annealing at 65°C for 30 s, a total of 35 cycles, (iii) extension at 72°C for 2 min, (iv) after 72°C Extend for 5min.

[0055] The results show ...

Embodiment 2

[0056] Example 2 Optimization of PCR reaction conditions

[0057] PCR amplification was carried out with primer 3 obtained by screening in Example 1. The PCR reaction system was the same as that in Example 1. The annealing temperature was adjusted between 50°C and 65°C and the number of cycles was adjusted between 30 and 45. The results showed that: GRAF -3 / GRAR-3 is the best annealing temperature for primer PCR at 65℃ ( figure 2 ); The number of cycles is 35.

Embodiment 3

[0058] Example 3 Composition of the kit

[0059] The kit composition of this embodiment includes: a sample DNA extraction solution, a PCR reaction solution and a positive control.

[0060] The sample DNA extraction solution here includes blood and semen DNA extraction solutions packaged independently; tissue DNA extraction solutions and water samples, and stool DNA extraction solutions.

[0061] Among them, the composition of blood and semen DNA extract is: 250 mL red blood cell lysate (10 mL DDT) for 50 reactions, 15 mL Buffer GA, 15 mL Buffer GB, 13 mL Buffer GD, 15 mL Buffer PW, 15 mL Buffer TE, 1 mL proteinase K, 10 mL 96% to 100% alcohol.

[0062] The composition of the tissue DNA extraction solution is: 50 mL nuclear lysate for 50 reactions, 30 mL 0.5M EDTA, 50 mL Wizard @ SV Lysis Buffer, 185mL column cleaning solution, 25mL RNase A solution, 2×25mL nuclease-free water.

[0063] The composition of water sample and stool DNA extraction solution is: 140 mL of ASL Buffer for 50 r...

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Abstract

The invention relates to the technical field of preparation of disease detection reagents and discloses a specific detection primer for swine toxoplasmosis and a detection kit. A dense granular protein 14 (GRA14) gene is taken as a target gene for diagnosis of the swine toxoplasmosis, specific detection primer GRAF-3 and GRAR-3 for toxoplasmosis are designed and screened out, and the sequence of each of the GRAF-3 and GRAR-3 is as shown as in SEQ ID No:1 and SEQ ID No.2. The toxoplasmosis is detected through the primer which is highly specific and highly flexibly, the result of a PCR (polymerase chain reaction) is easy to judge, epidemiological investigation for the toxoplasmosis and detection and clinical diagnosis of invisible infection can be realized by the aid the detection kit for the toxoplasmosis, prepared from the primer, and a foundation is laid for further research on diagnostic and preventive techniques and the like of the toxoplasmosis.

Description

Technical field [0001] The present invention relates to the technical field of preparation of disease detection reagents, and more specifically, to a specific detection primer and a detection kit for swine toxoplasmosis. Background technique [0002] Toxoplasmosis is a very serious zoonotic parasitic disease caused by Toxoplasma gondii infecting humans and various animals. Toxoplasmosis is widely distributed in my country and all over the world. The average human infection rate in Western countries is as high as about 30%. The average human infection rate in my country is 7.88%, and there are about 100 million people infected across the country, and the animal infection rate is even higher. Toxoplasmosis is not only one of the most common infectious diseases in humans, but also one of the main causes of death in people with immunosuppression and immunodeficiency. It is also closely related to prenatal and postnatal care. Pregnant women may directly affect the development of the fe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/90
CPCC12Q1/6893
Inventor 林瑞庆何茜翁亚彪吕梦娜黎盛琴唐陶张小刚胡伟高周志东
Owner SOUTH CHINA AGRI UNIV
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