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PCR (Polymerase Chain Reaction) primer, reagent or kit and identification method for identifying or assisting in identifying freshwater fish species

A technology for assisting identification and kits, which can be used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of difficult to obtain effective species-specific primers, etc. The effect of sample collection to improve feasibility

Pending Publication Date: 2021-10-29
SHANGHAI OCEAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using the PCR primers for PCR amplification of eDNA in water, based on high-throughput sequencing methods, can effectively solve the problem that it is difficult to obtain effective species-specific primers under the condition that there are many related species in water. Based on environmental DNA monitoring technology to provide effective technical support for fish taxonomy research and its diversity protection

Method used

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  • PCR (Polymerase Chain Reaction) primer, reagent or kit and identification method for identifying or assisting in identifying freshwater fish species
  • PCR (Polymerase Chain Reaction) primer, reagent or kit and identification method for identifying or assisting in identifying freshwater fish species
  • PCR (Polymerase Chain Reaction) primer, reagent or kit and identification method for identifying or assisting in identifying freshwater fish species

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Verification of fish universal primers, universal primer 16s 200 successfully amplified 29 common fish DNA

[0036] Muscle samples of 29 common fish species were collected, and DNA was extracted using the Animal Genomic DNA Rapid Extraction Kit of Shanghai Sangon Bioengineering Co., Ltd., and the extracted DNA was dissolved in TE buffer and stored at -20°C. Using the synthesized primer 16s 200, 29 kinds of fish DNA were amplified by PCR. The sample reaction system is 25 μL: 2.5 μL 10×ExTaqBuffer (Mg 2+ Plus) (20mM), 2μL dNTPMixture (2.5mM each), 1μL (10μmol / L) of upstream and downstream primers, 0.2μL TaKaRaTaqHS, 2μL DNA template, 16.3μL ddH 2 O. Reaction conditions: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing for 40 s, extension at 72°C for 1 min, 35 cycles; extension at 71°C for 10 min. PCR products were detected by 1% gel electrophoresis.

[0037] The amplification results of the fish samples with primer 16s 200 showed that brigh...

Embodiment 2

[0039] Using 16s 200 primers, 27 fish species distributed in Gehu Lake, Jiangsu Province, China were detected

[0040] We conducted fieldwork and sampling at Gehu Lake, the second largest lake after Taihu Lake in southern Jiangsu Province, China. Gehu Lake (119°44′15″-119°52′56″E, 31°28′19″-31°43′04″N) is located in the southwest of Changzhou City, Jiangsu Province, connected to the Yangtze River in the north, Taihu Lake in the east and connected to Changdang Lake is connected to Dongjiu and Xijiu in Yixing City in the south. The main rivers entering the lake are Biandan, Xiaxi, Huangli, Beigan and Zhonggan Rivers, etc., belonging to the Taihu Lake system. Lake Ge is narrow and long from north to north, shaped like a long eggplant, 25km long, 6.6km wide, and has a water surface area of ​​164km 2 , water storage capacity 1.74×10 8 m 3 , with an average water depth of 1.27m, is a typical shallow lake with multiple uses such as drinking, irrigation, sightseeing, and aquacultur...

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Abstract

The invention provides a novel PCR (Polymerase Chain Reaction) primer (16s 200) for identifying or assisting in identifying freshwater fish species. A nucleotide sequence of the primer is SEQ ID NO.1 in a sequence table. The PCR primer is applied to eDNA metabolism coding of a fish mitochondrial DNA sequence. The PCR primer is adopted to perform PCR amplification on eDNA in a water body, based on a high-throughput sequencing method, the problem that an effective species specific primer is difficult to obtain under the condition that various closely-related species exist in the water body can be effectively solved, and based on an environmental DNA monitoring technology, an effective technical support is provided for taxonomy research and diversity protection of fishes. The method for identifying the freshwater fish species by using the PCR primer has the advantages of short consumed time, simplicity and convenience in operation, high specificity and the like.

Description

technical field [0001] The invention relates to PCR primers, reagents or kits and identification methods for identification or auxiliary identification of freshwater fish species, belonging to the field of molecular ecology. Background technique [0002] Global biodiversity is rapidly decreasing due to environmental changes and human activities. Freshwater ecosystems are one of the most fragile ecosystems on earth. Freshwater fish are an important part of freshwater ecosystems. Due to factors such as climate change, habitat degradation, biological invasion, and overfishing, the diversity of freshwater fish is rapidly decreasing. . The ability to accurately assess the responses of fish populations and aquatic communities to environmental changes and quantify the impact of changes in fish community structure and function on aquatic ecosystems is critical for the conservation of freshwater ecosystems and fish diversity. The traditional fish monitoring technology mainly uses n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2527/101C12Q2527/107C12Q2527/125
Inventor 胡文静苏超群张真刘其根胡忠军
Owner SHANGHAI OCEAN UNIV
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