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Specific primer pair for identifying spotted ampullaria gigas in assisted manner and application of specific primer pair

A technology of specific primer pair and auxiliary identification, applied in the biological field, can solve problems such as difficulty in distinguishing snail species by morphology and color, variation in morphology and color characteristics of snail species, uncertainty, etc., to achieve rapid and efficient identification, easy operation, and required low cost effect

Inactive Publication Date: 2016-07-13
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high environmental adaptability of apple snails and the influence of factors such as food sources, external environment, and developmental stages, the variation of morphological and color characteristics within snail species is very easy to occur, so it is very difficult and uncertain to distinguish snail species from the shape and color. sex

Method used

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  • Specific primer pair for identifying spotted ampullaria gigas in assisted manner and application of specific primer pair
  • Specific primer pair for identifying spotted ampullaria gigas in assisted manner and application of specific primer pair
  • Specific primer pair for identifying spotted ampullaria gigas in assisted manner and application of specific primer pair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Design of specific primer pairs

[0046] On the basis of obtaining two kinds of foreign invading snail mitochondrial COI gene DNA barcode segments, the present invention designs specific primer pairs for snail snails through multiple sequence comparison analysis.

[0047] Based on the common mitochondrial COI segment of the snail snails of the genus snails, a specific primer pair can be designed and synthesized to identify snails:

[0048] PmacF (sequence 1 in the sequence list): 5’-TTTAGTTGGGGCTGGTTTGAGTTTG-3’; Tm=62.8°C, GC%=44%;

[0049] PmacR (sequence 2 in the sequence listing): 5’-AGCTAAAGGAGGGTACACTGTTCAC-3’; Tm=60.3°C, GC%=48%.

Embodiment 2

[0050] Example 2: Application of specific primers for detection of Phalaenopsis punctatus

[0051] In the embodiment, the following experiments are carried out for samples 1-24:

[0052] 1. Extraction of genomic DNA: use kit method to extract genomic DNA of single egg. The samples to be tested included two kinds of snail samples with a total of 24 samples. Genomic DNA extraction is based on the blood / cell / tissue genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. The steps and reagent dosage are slightly changed. The specific steps are:

[0053] (1) Take a single Fushou snail egg, wash the surface with 75% alcohol and wash twice with distilled water, dry it on clean filter paper, and transfer it to a 1.5ml PCR tube;

[0054] (2) Add 180μl of GA buffer to the centrifuge tube, and break the egg shell with a pipette tip to release the tissue fluid in the egg

[0055] (3) Add 20μl of ProK (20mg / ml), shake and mix well, and bath at 56℃ for 1h;

[0056] (4) Add 2...

example 3

[0071] Example 3: Detection of P. snail samples at different developmental stages

[0072] The samples used for the detection of samples of P. snails at different developmental stages include samples 25-27, 3 samples in total, and the following experiments are performed:

[0073] 1. Genomic DNA extraction

[0074] Sampling single-grained eggs in book 25, single-headed snails in sample 26, carefully remove the snail shell to retain the tissue in the shell, and 100 mg of gastropod tissue in sample 27, respectively, washed in 75% absolute ethanol and washed twice with distilled water. After drying on the clean filter paper, transfer to a 1.5ml centrifuge tube. The other steps are the same as in Example 2.

[0075] 2. PCR amplification with specific primers for P. sibiricus at various developmental stages

[0076] Using the genomic DNA of step 1 as a template and sterile water as a negative control, PCR amplification was performed using the specific primer pair designed in Example 1 to ob...

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Abstract

The invention discloses a specific primer pair for identifying spotted ampullaria gigas in an assisted manner and application of the specific primer pair.The specific primer pair provided by the invention consists of a DNA sequence shown in a sequence 1 of a sequence table and a DNA sequence shown in a sequence 2 of the sequence table.By the specific primer pair disclosed by the invention, identification of the spotted ampullaria gigas can be carried out in the assisted manner from the molecular level, and is not affected by individual development states of specimens or integrity of samples, and the specific primer pair has the advantages of high efficiency, convenience and accuracy.The problem of type identification of the spotted ampullaria gigas which perplexes plant protection and port quarantine inspection departments for a long time is solved.The specific primer pair can be widely used for plant protection departments, quarantine departments and institutions for researching ampullaria gigas.

Description

technical field [0001] The invention relates to a group of specific primer pairs for auxiliary identification of spotted apple snails and the application thereof, belonging to the field of biotechnology. Background technique [0002] Apple snails, also known as big bottle snails and apple snails (Goldenapplesnail), are native to the Amazon basin in South America; they belong to the mollusca (Mollusca), gastropod (Gastropoda), new gastropod (Caeogastropoda), bottle snail family (Ampullariidae) ), Apple snails ( Pomacea Perry, 1810). Apple snails are recognized worldwide as vicious invasive aquatic organisms, and have invaded East Asia, Southeast Asia, South Asia and North America and caused serious damage. Since the apple snail has the characteristics of wide feeding habits, strong reproductive ability, high hatching rate, short maturity cycle, strong adaptability and tolerance, and lack of natural enemies, it has rapidly expanded and colonized in many areas since it was in...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888
Inventor 俞晓平杨倩倩刘苏汶刘光富许益鹏张蓬军蒋家成
Owner CHINA JILIANG UNIV
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