Amplification primer and method of nassariidae mitochondria COI gene

A technology for amplification primers and Heliaceae, which is applied in the field of molecular biology, can solve the problems of low amplification efficiency of COI general primers and high variability of COI sequences, and achieve the goal of improving amplification efficiency and correctness, and high-efficiency amplification Effect

Inactive Publication Date: 2015-03-04
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0006] Aiming at the problems of high variability of COI sequences of Spirulinaceae species and low amplification efficiency of COI general primers, the present invention obtains a pair of amplification primers of Spirulinaceae mitochondrial COI gene through a large number of experiments. Based on the primers, it provides the amplification method of the Mitochondrial COI gene and the detection method of the Heliaceae species

Method used

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Examples

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Effect test

Embodiment 1

[0018] 1. Sample collection: From 2008 to 2012, a total of 150 individuals of 15 species of Snail family were collected from the north and south coasts of China.

[0019] 2. DNA extraction: DNA extraction was performed on the collected samples by CTAB method.

[0020] 3. COI universal amplification primer amplification: use the COI universal amplification primer LCO1490F designed by Folmer et al. 150 individuals were subjected to PCR amplification. The annealing temperature of PCR thermal cycle is different for different species, and the annealing temperature range is 43 oC ~50 oC . After the amplified product was detected by 1.5 times agarose electrophoresis, it was sent to a sequencing company for bidirectional sequencing. Finally, the COI sequences of 35 individuals from 4 species were obtained, the length of which was about 678bp, and the remaining individuals were not amplified.

[0021] 4. Download the sequence: Download the Spirulina species from Genban...

Embodiment 2

[0028] From 2008 to 2012, the Snail family species in the southeast coast were collected separately: Snail snail ( Nassarius rutilans ), longitudinal rib textured screw ( Nassarius variciferus ), checkered textured screw ( Nassarius clathratus ), Sig textured screw ( Nassarius siquinjorensis ), half-pleated textured screw ( Nassarius semiplicatus ) 5 species of larvae and adults, a total of 25 individuals, the phenol-chloroform method was used to extract DNA from the collected samples, and a total of 25 samples were obtained, including 5 larvae. Use the specific primer pair ZLCO1490 obtained in Example 1 to amplify 25 DNA samples, the amplification conditions are: 10 μL PCR reaction system: 1 μL 10×buffer Mg 2+ , 1 μL DNTP (2 mM), 0.6 μL Mgcl 2(25mM), 1μL primer MLCO1490F (10μM), 1μL primer MHCO2198R (10μM), 0.08μL r-Taq (5u / μL), 1μL DNA template, 4.32μL double distilled water, the annealing temperature of the PCR thermal cycle is 49 o c. The amplified products were...

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Abstract

The invention belongs to the field of molecular biology, and provides an amplification primer of a nassariidae mitochondria COI gene, and a method for amplifying the nassariidae mitochondria COI gene by using the primer. According to the amplification primer of the nassariidae mitochondria COI gene, the sequences are ZLCO1490F:5'-TTTCTACAAATCATAAAGACATTGG-3' and ZHCO2198R:5'-TATACCTCCGGGTGACCAAAAAATCA-3'. The amplification primer of the nassariidae mitochondria COI gene provided by the invention is capable of effectively amplifying COI sequences of different species of the nassariidae, and has great significance in research of species classification, system development, systematic geography, toxin tolerance and protection of nassariidae genetic resources by using the COI sequences.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a snail family mitochondrial COI [cytochrome c Oxidase subunit I (cytochrome c oxidase subunit I)] gene amplification primers, using the primers to amplify the method for the mitochondrial COI gene of the Heliaceae. [0002] Background technique [0003] Snail family ( Nassarius ) belongs to Gastropoda ( Gastropoda ), Probranchia ( Prosobranchia ), Neogastropods ( Neogastropoda ), which are distributed in seas all over the world. There are many species, which play an important role in maintaining the ecological balance of the intertidal zone of the ocean. Some species can also be used as an indicator tool for the detection of marine toxic pollutant tributyltin (TBT). Most of the snail species have delicious meat and are widely loved by Chinese consumers and have important economic value. However, in recent years, incidents of poisoning due to eating snails have been repo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888
Inventor 邹山梅王长海
Owner NANJING AGRICULTURAL UNIVERSITY
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