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An environmental DNA method to detect the distribution of giant salamanders

A giant salamander and environmental technology, applied in the molecular field, can solve the problems of small number of natural populations, ineffective survey methods, time-consuming and labor-intensive costs, etc., and achieve the effect of high amplification efficiency.

Active Publication Date: 2022-05-31
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, due to the fact that this species lives underwater, its activities are secretive, and its natural population is very small, traditional survey methods are ineffective, time-consuming, labor-intensive, and costly, and it is urgent to develop new technical means

Method used

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  • An environmental DNA method to detect the distribution of giant salamanders
  • An environmental DNA method to detect the distribution of giant salamanders
  • An environmental DNA method to detect the distribution of giant salamanders

Examples

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Effect test

Embodiment 1

[0025] Example 1: Primer Sensitivity Verification

[0026] 1. Chinese giant salamander tissue sample DNA was extracted using TIANamp Genomic DNA Kit. DNA from tissue samples of 22 Chinese giant salamanders captured from Gutian Mountain, Zhejiang Province (concentration: 1.55 ng / μL, numbers 1-22) were amplified by PCR using the 5 pairs of primers described above. Amplification results showed only clear band after agarose gel electrophoresis, and the sequence length of the band was between 150-250bp.

[0027] According to the brightness of the amplified bands, that is, the amplification efficiency, it was judged that the amplification effects of the primers Ad_Cytb_E1, Ad_Cytb_E3, Ad_Cytb_E5 were better than those of Ad_Cytb_E2, Ad_Cytb_E4.

[0028] 2. Select 6 DNA samples with slightly poor amplification effects, dilute the DNA stock solution 20 times (sample concentration 0.0775ng / μL), and use the above-mentioned 3 pairs of primers with better amplification effects to carry o...

Embodiment 2

[0031] Example 2: Primer Specificity Verification

[0032] Two methods were used for primer specificity verification:

[0033] (1) On the NCBI website, use the primer BLAST function (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to perform simulation verification of the Ad_Cytb_E1 primer specificity against the GenBank database. Ad_Cytb_E1 was proved by primer BLAST: the specificity is high, and the 202 sequences that completely match it are all from the Chinese giant salamander, and the sequences from other species cannot be completely matched, and the number of mismatched bases is 4 to 10, and the giant salamander is closely related The species Japanese giant salamander had a sequence mismatch of 1 base, indicating that it might also be possible to amplify this sample.

[0034] (2) The Chinese giant salamander and 7 other amphibians in the same area: Chinese salamander (Paramesotritonchinesis), Tibetan mountain stream salamander (Batrachuperus tibetanus), light-shoulder...

Embodiment 3

[0038] Example 3: Verification of Primer Versatility

[0039] 1. To test the amplification of each branch of Chinese giant salamander with Ad_Cytb_E1 primer. The test method is as follows: select 7 representative individuals from the 4 genetic branches of Chinese giant salamander (as shown in Table 2), extract the DNA of these 7 individuals, use Ad_Cytb_E1 primers for PCR amplification, and all can be seen after gel electrophoresis The target band was verified to be the cytochrome b gene sequence of Chinese giant salamander by next-generation sequencing, which showed that the Ad_Cytb_E1 primer had good amplification efficiency for different genetic lines of Chinese giant salamander.

[0040] Table 2 Sample information of giant salamander from different clades

[0041]

[0042] 2. In order to further verify whether the environmental DNA of different clades of giant salamander can be simultaneously amplified, next-generation sequencing is carried out. The DNA of each sample i...

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Abstract

The invention belongs to the field of molecular technology, and in particular relates to an environmental DNA method for detecting the distribution of giant salamanders. The specific technical plan is: an environmental DNA method for detecting the distribution of giant salamanders. At least one pair of primers is used to perform PCR amplification detection on the sample to be tested. If a positive band of 158 to 224 bp appears in the electrophoresis result, it is considered that the source of the sample to be tested is from the environment. Giant salamanders are distributed, the primer pair is Ad_Cytb_E1, and the sequences are: SEQ ID NO:1, SEQ ID NO:2. The present invention uses environmental DNA such as Chinese giant salamander feces, skin exfoliation and corpses in the residual water body, and combines molecular biology techniques to develop primers with high specificity, high sensitivity and strong versatility, and establishes a primer that can target giant salamanders. A method for rapid, standardized detection of the distribution of different species.

Description

technical field [0001] The invention belongs to the field of molecular technology, and in particular relates to an environmental DNA method for detecting the distribution of giant salamanders. Background technique [0002] The Chinese giant salamander (Andrias davidianus complex) is the flagship species of global amphibian conservation. Due to overuse, habitat quality decline, area reduction, environmental pollution and other reasons, its wild population has declined sharply. The Alliance Red List lists it as a critically endangered species. Habitat protection and artificial population enhancement and release are important protection measures for this species, and detection of original or released individuals and their population distribution is the basis for formulating and evaluating protection measures. [0003] However, due to the fact that this species lives underwater, its activities are secretive, and its natural population is very small, traditional survey methods a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2565/125Y02A50/30
Inventor 王杰谢锋江建平刘萍
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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