Neospora caninum specific nest-polymerase chain reaction (PCR) test kit and preparation method

A technology of Neospora and a kit is applied in the preparation of the above-mentioned nest-PCR detection kit and the field of Neospora-specific nest-PCR detection kits, which can solve the problem of inability to meet actual needs, and backwardness in the quarantine and diagnosis of Neospora disease, etc. problems, to achieve social significance, easy operation, high specificity

Inactive Publication Date: 2013-01-23
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The quarantine diagnosis of neosporosis at home and abroad is still relatively backward, and the construction of many domestic laboratories is still blank. The detection of the disease still uses ELISA, IFTA and PCR, which is far from meeting the actual needs.

Method used

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  • Neospora caninum specific nest-polymerase chain reaction (PCR) test kit and preparation method
  • Neospora caninum specific nest-polymerase chain reaction (PCR) test kit and preparation method
  • Neospora caninum specific nest-polymerase chain reaction (PCR) test kit and preparation method

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Experimental program
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Effect test

Embodiment 1

[0052] According to the principle of nest-PCR amplification, the present invention designs and synthesizes a group of nest-PCR primers for specifically amplifying Neospora gene, and entrusts Shanghai Biological Engineering Co., Ltd. to synthesize the primers. After primer synthesis, use sterilized distilled water to make 100mM stock solution (stock solution), and then use ddH 2 O diluted to 10mM as a working solution. The primer sequences are as follows:

[0053] Primers for the first round of PCR:

[0054] OF: 5'-AAACCAGGAAAACAAATGA-3'

[0055] OR: 5'-TTGACGGTGTCTGAAAATC-3'

[0056] Primers for the second round of PCR:

[0057] IF: 5'-ACGGTCACATTGTTCATCTA-3'

[0058] IR: 5'-AAGTCCTGGGTATTGTTATTG-3

Embodiment 2

[0060] Kit assembly:

[0061] 1) 10×PCR reaction solution: 300mM dNTPs (purchased from Japan TAKARA company) 20μl, Tris (purchased from US Pragma company) 2.42mg, KCl (purchased from US Sigma company) 7.45mg, MgCl 2 (purchased from Sigma, USA) 0.258 mg, 200 U of Taq enzyme (purchased from TAKARA, Japan) 20 μl, add 150 μl ddH 2 O, adjust the pH to 8.5 with HCl, use ddH 2 O was adjusted to 200 μl;

[0062] 2) Preparation of positive control DNA: the positive control DNA is Neospora genomic DNA, and the cultured Neospora are counted with a cell counting plate, and 10 6 Genomic DNA was extracted from each Neospora and diluted to 100 μl.

[0063] 3) Kit assembly: each kit is for testing 100 samples:

[0064] Assemble the kit as follows:

[0065] To detect 100 samples per kit, assemble the kit according to the following content (the entire operation requires a sterile environment):

[0066] 10×PCR reaction solution 200μl,

[0067] 10mM primer OF 50μl;

[0068] 10mM primer...

Embodiment 3

[0074] The using method of kit of the present invention is as follows:

[0075] 1), the first round of PCR amplification:

[0076] After the genomic DNA is extracted from the sample to be tested, the reagents in this kit are used for the first round of PCR amplification. The PCR reaction loading system is as follows, the total reaction system is 20 μl (the amount of sample DNA is determined according to the concentration of the extracted genomic DNA, and finally with ddH 2 O to make up 20 μl; positive control volume is 1 μl):

[0077] 10×PCR reaction solution: 2μl;

[0078] Determine the sample DNA according to the amount of extracted DNA:

[0079] Primer OF (10mM): 1 μl;

[0080] Primer OR (10mM): 1μl;

[0081] ddH2O: Make up to 20 μl.

[0082] Add the negative and positive controls to the marked tubes respectively, then add the samples one by one and mark them well, and place them in the PCR instrument; the conditions of the first round of PCR are: pre-denaturation at ...

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Abstract

The invention provides a neospora caninum specific nest-polymerase chain reaction (PCR), which is used for testing tachyzoite, tissue cyst and oocyst infected with neospora caninum in different animals. The invention also discloses a preparation method of the nest-PCR test kit. The nest-PCR is used for amplifying some specific molecular fragment of a gene of a neospora caninum for diagnosing neosporosis. The invention relates to neospora caninum and a neospora caninum specific gene SEQNo.1 which is screened from an ultramicro-structure suppressed subtractive hybridization library. The gene is the specific to neospora caninum, so amplification of fragments of SEQNo.1 gene in animal tissue and dung by the nest-PCR can be used for molecule detection of neospora caninum and diagnosing acute, chronic and recessive infection with neospora caninum.

Description

technical field [0001] The invention provides a Neospora-specific nest-PCR detection kit, which is used for the detection of tachyzoites, tissue cysts and oocysts infected by Neospora in different animals. The invention also discloses the above-mentioned nest-PCR detection kit The preparation method belongs to the technical field of molecular biology detection. Background technique [0002] Neospora ( Neospora caninum ) as an Apicomplexan parasite, phylogenetic analysis of ribosomal DNA showed that it was related to Toxoplasma gondii, Hammondia heydorni Relatively close in kinship, the general experimental detection methods are mainly serological methods, including IFAT, ELISA, PCR, etc., because they are different from the expression of antigens in different stages of parasites and the production of antibodies, so ELISA and IFTA Infection cannot be detected at all stages of the parasite's life cycle, especially in the definitive host and sexual stages. Genomic DNA is ve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/30C12N15/11C12Q1/68C12Q1/04
Inventor 李建华张西臣贺鹏飞宫鹏涛吕强李赫杨举王伟利张国才任文陟
Owner JILIN UNIV
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