Primers, primer kit and identification method for identifying pseudobagrus ussuriensis, pseudobagrus tenuis and reciprocal hybrid individuals of pseudobagrus ussuriensis and pseudobagrus tenuis
An identification method, Ussuri's technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as difficulty in distinguishing, achieve accurate identification results, save labor, and improve efficiency and the effect of accuracy
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Embodiment 1
[0025] Example 1 The composition and preparation of the PCR system for individual identification of Pseudomonas Ussuri, Pseudomonas chinensis and their positive and negative hybridization
[0026] (1) PCR system composition:
[0027] dNTPs (10 mM), Taq DNA polymerase (5 U / µL), and 10×PCR Buffer are all products of Jiangsu Kangwei Century Biotechnology Co., Ltd.;
[0028] Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.: PCR primers were carried out using the nucleotide sequences SEQ ID NO: 1-4 of the 4 interspecies-specific sites of P. Design, obtain nucleotide sequence as four pairs of primers of SEQ ID NO:5-12:
[0029] SEQ5: 5'-CTCTTGCCTATATCGTGCCG-3'
[0030] SEQ6: 5'-GTAATCATACTGCTTCTTGGGT-3'
[0031] SEQ7: 5'-GTATTTATTGGAGGATGAGGA-3'
[0032] SEQ8: 5'-GTTATGGAGTAGCACAGTCGT-3'
[0033] SEQ9: 5'-TCCTACACTTTCATCTCCCAG-3'
[0034] SEQ10: 5'-ACACTTCAACAGCCGCATC-3'
[0035] SEQ11: 5'-GCTTTGCCGTTACGATACTT-3'
[0036] SEQ 12: 5'-TGCCTGAATCTGAACTGACT...
Embodiment 2
[0044] Example 2 Evaluation of the identification effect in the known samples of Ussuri pufferfish, puffertail pufferfish and their positive and negative hybridization samples
[0045] S1. Randomly select 80 P. ussurii and 80 P. spp. from the breeding farm, artificially induce labor and hybridize P. spp. ♀×P. There were 80 fish ♂ each, and a small amount of caudal fin tissue was cut from each sample, and the genomic DNA was extracted by the phenol-chloroform method, and the DNA concentration was diluted to 80-100 ng / µL with sterilized ultrapure water.
[0046] S2. Using the genomic DNA extracted from S1 as a template, prepare PCR reaction mixtures for 4 primer pairs according to the system in (3) in Example 1.
[0047] S3. Amplify on PCR instrument, pre-denaturation at 95°C for 6-8 minutes; denaturation at 95°C for 45-60 seconds, annealing at 50-64°C for 45-60 seconds, extension at 72°C for 45-60 seconds, 30 -37 cycles; final extension at 72°C for 15-20 minutes, and store PCR...
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