Mussel environment DNA macro bar code primer, identification method thereof, and application of primer and identification method

A technology of macro-barcode and identification method, which is applied in mussel environmental DNA macro-barcode primer and identification field, can solve the problems of poor practicality, limited application, and insufficient number of species, etc., and achieve the improvement of sensitivity and accuracy, versatility and applicability Good performance, saving manpower and financial resources

Active Publication Date: 2021-10-19
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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  • Mussel environment DNA macro bar code primer, identification method thereof, and application of primer and identification method
  • Mussel environment DNA macro bar code primer, identification method thereof, and application of primer and identification method
  • Mussel environment DNA macro bar code primer, identification method thereof, and application of primer and identification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0048] 1) Select sequence and design primers

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

[0050] Table 1 Primer information designed by this patent

[0051]

[0052] 2) Ordinary PCR amplification screening primers

[0053] Extract the tissue DNA of a single mussel species (a total of 32 species, which are accumulated samples in the laboratory, see Table 2 for details): take the adductor muscle of the sample fixed in alcohol or the sample stored at -80°C, digest it with proteinase K, and use the kit ( Genomic DNA Purification System), and finally dissolved in 100 μl sterile double distilled water. The 30 species of mussels were tested by PCR with the designed universal primers. The PCR reaction conditions were: 95°C pre-denaturation fo...

Embodiment 2

[0076] Example 2: Research on mussel diversity in Poyang Lake based on environmental DNA metabarcode

[0077] 1) Environmental DNA sample collection and filtration

[0078] Sample collection was carried out in the waters of Poyang Lake in April 2019. A total of 4 sampling areas (including Tongjiang waterway, main lake area, Junshan Lake and Qinglan Lake) and 20 sampling points were set up. For the sampling map, see image 3 , use a water collector to collect 1L water samples at about 0.5-1m underwater, do 3 biological repetitions, store the water samples in sterile jars at low temperature, and use a 0.45μm mixed cellulose filter membrane within 24 hours Filtration, before and after each filtration, the filter material is disinfected to avoid cross-contamination. One negative control was set for each filtration. While collecting water samples, the traditional method was used to collect mussels with homemade mussel rakes, and the morphological characteristics were used to iden...

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Abstract

The invention particularly relates to a mussel environment DNA macro bar code primer, an identification method thereof, and application of the primer and identification method, and belongs to the field of molecular ecology. The mussel environment DNA macro bar code primer is named 16SPP300 or 16SPP147; the primer 16SPP300 is composed of an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO: 2; and the primer 16SPP147 is composed of an upstream primer as shown in SEQ ID NO: 3 and a downstream primer as shown in SEQ ID NO: 4. The invention further discloses the identification method of environment DNA macro bar code for mussel community structure research, and application thereof. According to the method, abundance data of mussel animals which are difficult to collect and endangered in a traditional investigation method can be supplemented, male mussel species can be identified, the identification level, the species detection rate and the abundance evaluation accuracy of the mussels are effectively improved, and the method is an effective and reliable new mussel community structure investigation method and 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, identification method and application. Background technique [0002] Mussels belong to the phylum Mollusca, class Bivalvia, and order Unionoida, and are widely distributed in North America, Eurasia, Central America, Africa and Southeast Asia. Mussels are macrobenthic animals and one of the most important biological groups in ecosystems. As part of the food chain they are food for small animals such as demersal 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. If the freshwater bivalves in lakes or rivers disappear, it will cause a series of ecological problems. In recent decades, the global mussel population density and species richness have declined significantly, and the rate of decline in the number of s...

Claims

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Application Information

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Patent Type & Authority Applications(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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