A kind of mussel environmental DNA metabarcoding primer, identification method and application

A technology of metabarcode and identification method, which is applied in the field of identification and mussel environmental DNA macrobarcode primers, which can solve the problems of poor practicability, limited application, and insufficient number of species, etc., to achieve improved sensitivity and accuracy, versatility and applicability Good performance, saving manpower and financial resources

Active Publication Date: 2022-08-09
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The number of species currently used for the monitoring of mussel environmental DNA metabarcoding primers is not enough and the practicability is poor, which limits its application

Method used

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  • A kind of mussel environmental DNA metabarcoding primer, identification method and application
  • A kind of mussel environmental DNA metabarcoding primer, identification method and application
  • A kind of mussel environmental DNA metabarcoding primer, identification method and application

Examples

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Effect test

Embodiment 1

[0047] Example 1: Screening of the most ideal mussel environmental DNA metabarcoding universal primers

[0048] 1) Select sequences and design primers

[0049] Select the full sequence segment of mitochondrial 16SrRNA gene, and design the product as 2 pairs of universal primers of 100-300bp. The primer information is shown in Table 1:

[0050] Table 1 Information of primers designed in this patent

[0051]

[0052] 2) Common PCR amplification screening primers

[0053] Extract tissue DNA from a single mussel species (32 species in total, which are samples accumulated in the laboratory, see Table 2 for details): Take the adductor muscle of alcohol-fixed samples or samples stored at -80°C, digested with proteinase K, and the kit ( SV Genomic DNAPurification System) and finally dissolved in 100 μl of sterile double distilled water. The 30 kinds of mussels were tested by PCR with the designed universal primers. The PCR reaction conditions were as follows: pre-denaturation a...

Embodiment 2

[0075] Example 2: Diversity of mussels in Poyang Lake based on environmental DNA metabarcoding

[0076] 1) Environmental DNA sample collection and filtration

[0077] In April 2019, samples were collected in the waters of Poyang Lake. A total of 4 sampling areas (including the Tongjiang waterway, the main lake area, Junshan Lake and Qinglan Lake) and 20 sampling points were set up. Please refer to the sampling map. image 3 , use a water collector to collect a 1L water sample at about 0.5-1m underwater, do 3 biological replicates, store the water sample in a sterile jar at low temperature, and use a 0.45μm mixed cellulose filter within 24 hours. Before and after each filtration, the filter equipment is disinfected to avoid cross-contamination. Set 1 negative control per filter. While collecting water samples, traditional methods were used to collect mussels using homemade mussel rakes, and morphological characteristics were used to identify mussel species. The water sample ...

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Abstract

The invention specifically relates to a mussel environmental DNA macrobarcode primer, an identification method and an application, and belongs to the field of molecular ecology. The invention discloses a mussel environmental DNA macrobarcode primer, and the name of the primer is 16SPP300 or 16SPP147; wherein, the primer 16SPP300 consists of an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO: 2; the primer 16SPP147 consists of the upstream primer shown in SEQ ID NO:3 and the downstream primer shown in SEQ ID NO:4. The invention also discloses an environmental DNA macrobarcoding identification method and application for the research of mussel community structure. The method of the invention can supplement the abundance data of mussels that are difficult to collect and endangered in traditional survey methods, and can identify male mussels, effectively improving the identification level, species detection rate and abundance evaluation accuracy of mussels. An effective and reliable new method for the investigation of mussel community structure, which has practical value.

Description

technical field [0001] The invention relates to the field of molecular ecology, in particular to a mussel environmental DNA macrobarcode primer, an identification method and an application. Background technique [0002] Mussels belong to the phylum Mollusca, Bivalvia, and Unionoida, and are widely distributed in North America, Eurasia, Central America, Africa and Southeast Asia. Mussels are macrobenthos and one of the most important biological groups in the ecosystem. As part of the food chain they are food for small animals such as bottom fish and some birds. As filter feeders, they maintain water quality by filtering nutrients, organic matter, and plankton. They are in a very critical position in the food web, and the disappearance of freshwater bivalves in lakes or rivers will cause a series of ecological problems. In recent decades, the global mussel population density and species richness have declined significantly, and the speed of the decline of some species has e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6869C12N15/11
CPCC12Q1/6888C12Q1/6869C12Q2531/113C12Q2535/122C12Q2537/165Y02A90/40Y02A40/81
Inventor 周春花陈金萍吴小平黄晓晨欧阳珊
Owner NANCHANG UNIV
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