Universal metabarcoding amplification primers for freshwater fish mitochondria 12S and application method thereof

A technology of macro-barcode and amplification primers, which is applied in the field of 12S universal macro-barcode amplification primers for freshwater fish mitochondria, which can solve the problems of low DNA concentration in free fish, difficult sequencing of primer amplification products, and poor amplification ability of fish communities problems such as low degeneracy, strong amplification preference, and high sensitivity

Active Publication Date: 2019-06-28
南京易基诺环保科技有限公司
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AI Technical Summary

Problems solved by technology

[0010] Aiming at the low concentration of free fish DNA in environmental samples and the poor amplification ability of existing COI primers for fish communities in environmental samples, the present invention provides a general-purpose macrobarcode amplification primer for mitochondrial 12S in freshwater fish. Mitochondria 12S is an animal A relatively conserved gene in the mitochondrial genome, one of the potential barcode regions for species identification
[0011] The present invention also provides a method for applying primers for the 12S universal macrobarcode amplification of freshwater fish mitochondria, which further solves the problem that the products amplified by existing primers are difficult to sequence on a high-throughput sequencing platform. 12S gene sequence has designed 6 upstream primers and 10 downstream primers. The combination of upstream and downstream primers can amplify PCR products ranging in length from 70bp to 350bp. The degeneracy of the primers is low, the success rate of PCR is high, and the PCR products The length is compatible with the second-generation high-throughput sequencing platform, and can be used for fish species identification, fish diversity analysis, etc.

Method used

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  • Universal metabarcoding amplification primers for freshwater fish mitochondria 12S and application method thereof
  • Universal metabarcoding amplification primers for freshwater fish mitochondria 12S and application method thereof
  • Universal metabarcoding amplification primers for freshwater fish mitochondria 12S and application method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0051] (1) List of common primer pairs for fish mitochondrial 12S gene metabarcode

[0052] Table 1 The size of the fragments amplified by the combination of upstream and downstream primers

[0053]

Embodiment 2

[0055] The fish samples were collected in Zhushan Bay (120.036° east longitude, 31.373° north latitude) and Miaogang (120.461° east longitude, 31.002° north latitude) in Taihu Lake, Jiangsu Province. Ground cages and gillnets were used to catch fish on site, and the collected fish samples were taken to the laboratory for species identification and separation. A total of 10 species of fish were collected (red-finned bream, bream, bream, bream, bighead carp, silver carp, common carp, crucian carp, large scale loach, sword anchovy), and the DNA of each fish sample was extracted separately. Fish-F1 and Fish-R1.1 (Fish-R1.2) primer combination, Fish-F2 and Fish-R2.1 (Fish-R2.2) primer combination, Fish-F3 and Fish-R3.1 ( Fish-R3.2) primer combination, Fish-F4 and Fish-R4 primer combination, Fish-F5 and Fish-R5 primer combination, Fish-F6 and Fish-R6.1 (Fish-R6.2) primer pair extraction For PCR amplification of fish DNA, the molar ratio of the combination of upstream primers and do...

Embodiment 3

[0059] In Zhushan Bay (120.036° east longitude, 31.373° north latitude) and Miaogang (120.461° east longitude, 31.002° north latitude), 0.5-1L of surface water was collected with a 1L sampling bottle, and the glass fiber filter with a pore size of 0.22μm was used in the laboratory. Membrane filtration to remove water, according to The Water DNA Kit (OMEGA, CHINA) standard operation was used to extract the environmental DNA retained in the filter membrane, and PCR amplification was performed on the filter membrane DNA using the mitochondrial 12S primers and reagents in Example 2. PCR amplification products were detected by 2% agarose electrophoresis ( figure 2 shown). The PCR product was recovered by tapping the gel through the gel recovery kit (MinElute Gel Extraction Kit), using Qubit TM dsDNA HS Assay Kits were used for accurate quantification, and the purified PCR products were analyzed for fragment size using Bioanalyser 2100 (Agilent Technologies, USA). The analysis ...

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Abstract

The invention discloses universal metabarcoding amplification primers for freshwater fish mitochondria 12S and an application method thereof, and belongs to the field of biotechnology. The 16 metabarcoding amplification primers are designed according to the gene sequences of common freshwater fish mitochondria 12S, including 6 upstream primers and 10 downstream primers; the primer pairs have widecoverage on fish communities and high amplification efficiency, the primer combinations can amplify products with the length of 70bp to 350bp and are compatible with different high-throughput sequencing platforms, the multi-primer combination method can reduce non-specific amplification and increase the PCR success rate, and the primers can be widely applied to fish species identification, fish diversity analysis and other research as characteristic barcode sequences.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a freshwater fish mitochondrial 12S universal macro barcode amplification primer and an application method thereof. Background technique [0002] Metabarcoding (Metabarcoding) is currently one of the most powerful species diversity analysis techniques, especially playing an important role in the monitoring of endangered and rare species. With the improvement of high-throughput sequencing throughput and the reduction of sequencing costs, metabarcoding technology has begun to be more and more used in the investigation of environment and biodiversity. Among them, environmental source DNA includes water sample DNA, sediment DNA, and biofilm DNA; biological source DNA refers to DNA extracted from biological tissues, such as skin and hair. The two strands of DNA are antiparallel structures. People usually refer to the hydroxyl (-OH) end of DNA as the 3' end, and the end of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6869C12N15/11
CPCY02A90/40
Inventor 杨江华张效伟
Owner 南京易基诺环保科技有限公司
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