Primer pair, reagent kit and method for distinguishing brachymystax lenok from Hucho taimen
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A technique of primer pair and leptospirosis, which is applied in biochemical equipment and methods, resistance to vector-borne diseases, measurement/inspection of microorganisms, etc., can solve problems such as difficult identification, loss of external morphological characteristics, and urgent identification
Active Publication Date: 2019-07-23
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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After further processing, the original external morphological characteristics are lost, making it more di...
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Embodiment 1
[0020] Embodiment 1, method establishment
[0021] 1. Sample genomic DNA extraction
[0022] Collect 5-10 scales of the sample fish, add 40 μl of lysate (Protease K 0.5mg / ml, Tris (pH8.0) 10mM, KCl 50mM, Tween 20 0.3% (w / v), NP-40 0.3% (w / v)), 55°C for 2h, 98°C for 10min to lyse, mix evenly after lysis, centrifuge at 1000-2000rpm for 1-2min, extract the supernatant, which is the crude genomic DNA extract.
[0023] 2. Primer design
[0024] One pair of specific primers were designed according to the ITS gene and the mitochondrial genome of the salmon and jerky salmon, and a total of 4 primers were designed. The primer sequences are as follows:
[0025] ITS-F: 5'-GTGCCTAACGGGGAAAGG-3' (SEQ ID NO.1)
[0026] ITS-R: 5'-AACATACAGCAAATGCCATCC-3' (SEQ ID NO.2)
[0027] mtDNA-F: 5'-TAAGCAAAATGGGCAAAACC-3' (SEQ ID NO.3)
[0028] mtDNA-R: 5'-GTTATTCCAAGCGCACCTTC-3' (SEQ ID NO.4)
[0029] The present invention compares the ITS gene of the salmon ITS on NCBI with the transcriptome ...
Embodiment 2
[0040] Embodiment 2, sample detection
[0041] Using the identification method of Example 1, collect 50 adult fishes identified as wild leprosy salmon and salmon salmon in different watersheds according to their morphology, and get 5-10 scales of each adult fish for identification. According to the comparison between the electrophoresis results and the provided standard spectra of salmon salmon and salmon salmon, a specific band with a size of 298bp appeared in all 50 samples identified as salmon salmon by morphology, indicating that the samples were salmon salmon samples; 50 Two specific bands with a size of 298bp and a size of 192bp appeared in one sample of jerky salmon according to morphological identification, indicating that the sample was jerky salmon. The method is very sensitive, and fish eggs, juvenile fish and adult fish can all be distinguished, and the accuracy rate can reach 100%.
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Abstract
The invention discloses a primer pair, reagent kit and method for distinguishing brachymystax lenok from Hucho taimen. The primer pair comprises a primer pair 1 and a primer pair 2, wherein the primerpair 1 comprises an upstream primer ITS-F and a downstream primer ITS-R, and the primer pair 2 comprises an upstream primer mtDNA-F and a downstream primer mtDNA-R; the sequence of the ITS-F is shownas SEQID NO.1, the sequence of the ITS-R is shown as SEQID NO.2, the sequence of the mtDNA-F is shown as SEQID NO.3, and the sequence of the mtDNA-R is shown as SEQID NO.4. A new method for distinguishing brachymystax lenok from Hucho taimen based on a molecular biology technique principle is adopted for the first time at home and abroad, 1 pair of specific primers is designed by using ITS genesand mitochondrial genome of the brachymystax lenok and the Hucho taimen, and a new method for identifying the brachymystax lenok and the Hucho taimen can be realized by a PCR reaction. The primer pair, the reagent kit and the method can be used for distinguishing the brachymystax lenok from the Hucho taimen complete individual and processed products thereof, and have the characteristics of being accurate, quick, objective, fair, economical and practical.
Description
technical field [0001] The invention relates to a pair of primers, a kit and a method for distinguishing salmon and salmon, and belongs to the technical field of molecular identification. Background technique [0002] Brachymystax lenok (pallas) and Hucho taimen (pallas) are rare and valuable cold-water fishes in my country, belonging to the genus Lepidoptera and Salmonidae of the order Salmoniformes and the family Salmonidae. fish genus. In 1998, the salmon and jellyfish were included in the Red Data Book of Endangered Animals in China, and the endangered level was Vulnerable. In 2004, the two species were included in the Red List of Species in China. In recent years, the resources of lobster and jellyfish have been declining, and it is hard to see them. Both salmon and jellyfish are cold-water fish with similar living conditions. In order to better protect and rationally develop the germplasm resources of leach salmon and jelly salmon, and accurately assess their resourc...
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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2565/125Y02A50/30
Inventor 佟广香唐国盘匡友谊尹家胜徐伟张庆渔
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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