A kind of Neospora specific PCR detection kit and preparation method
A detection kit, Neospora technology, applied in the preparation of the above-mentioned nest-PCR detection kit, Neospora-specific nest-PCR detection kit field, can solve the backwardness of Neospora quarantine diagnosis and can not meet the actual needs and other issues, to achieve the effect of great social significance, easy operation, and simple operation
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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 O...
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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