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LAMP (loop-mediated isothermal amplification) primer group for identifying common flies of fresh cherries and application of LAMP primer group

A primer set, flies technology, applied in the biological field, can solve the problems of small larvae, difficult morphological identification, expensive equipment and so on

Active Publication Date: 2022-07-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the small size of flies and the difficulty of distinguishing larvae, professional morphological knowledge reserves are required, making morphological identification very difficult
Although the existing PCR molecular identification technology can achieve fast and accurate identification, it requires expensive instruments and equipment, and the cost of identification is high, so it is not suitable for popularization and application in grassroots departments

Method used

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  • LAMP (loop-mediated isothermal amplification) primer group for identifying common flies of fresh cherries and application of LAMP primer group
  • LAMP (loop-mediated isothermal amplification) primer group for identifying common flies of fresh cherries and application of LAMP primer group
  • LAMP (loop-mediated isothermal amplification) primer group for identifying common flies of fresh cherries and application of LAMP primer group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Example 1. Preparation of primer sets and kits for identifying three common flies of fresh-eating cherries-Drosophila melanogaster, Drosophila tangerines, and Drosophila melanogaster

[0117] 1. Screening of primer sets for the identification of three common flies that eat fresh cherries

[0118] 1. Based on the full-length sequences of COI genes in the mitochondrial genome of three common flies that eat fresh cherries—Drosophila melanogaster, Drosophila melanogaster, and Drosophila melanogaster (nucleotide sequences such as Sequence 13, Sequence 14 and Sequence 14, respectively). Sequence 15) as the target gene, design and synthesize primer set SWD1, primer set SWD2, primer set SWD3 and primer set SWD4 for identifying Drosophila melanogaster; primer set OFF1, primer set for identifying Drosophila melanogaster OFF2, primer set OFF3, primer set OFF4, primer set OFF5; primer set ECFF1, primer set ECFF2, primer set ECFF3, primer set ECFF4, primer set ECFF5, and primer set ...

Embodiment 2

[0141] Application of the kit prepared in Example 2 and Example 1 in the identification of three common flies that eat fresh cherries

[0142] 1. Identification of three common flies that eat fresh cherries by agarose gel electrophoresis

[0143] 1. Use a blood / cell / tissue sample genomic DNA extraction kit to extract the genomic DNA of the flies to be tested to obtain the genomic DNA of the flies to be tested.

[0144] 2. Using the genomic DNA of the fly to be tested as a template, use primer set SWD4, primer set OFF1, and primer set ECFF4 to carry out loop-mediated isothermal amplification to obtain amplification products.

[0145] The reaction system was 25 μL, consisting of 12.5 μL Warm Start LAMP 2×Master Mix, 1 μL template, primer mix and sterile ultrapure water. The primer mixture is a mixture composed of each primer in the primer set SWD4, the primer set OFF1 or the primer set ECFF4. In the reaction system, the final concentration of outer primer F3 and outer primer B...

Embodiment 3

[0166] Example 3. Specificity experiment

[0167] The test fly 1 is the spotted wing Drosophila numbered as 1.

[0168] The test fly 2 is the spotted wing Drosophila numbered 2.

[0169] The flies to be tested 3 are B. tangerines numbered 3.

[0170] The flies to be tested 4 are B. tangerines numbered 4.

[0171] The flies to be tested 5 are B. tangerines numbered 5.

[0172] The flies to be tested 6 are B. tangerines numbered 6.

[0173] The test fly 7 is B. citrifolia numbered 7.

[0174] The test fly 8 is B. tangerines numbered 8.

[0175] The test fly 9 is B. tangerines numbered 9.

[0176] The test fly 10 is B. tangerines numbered 10.

[0177] The test fly 11 is B. tangerines numbered 11.

[0178] The test fly 12 is B. tangerines numbered 12.

[0179] The flies to be tested 13 are B. tangerines numbered 13.

[0180] The test fly 14 is the European cherry flies numbered 14.

[0181] The test fly 15 is Drosophila melanogaster numbered 15.

[0182] The test fly 16 ...

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) primer group for identifying three common flies of fresh cherries and application of the LAMP primer group. The LAMP primer group comprises a primer group SWD4, a primer group OFF1 and a primer group ECFF4; the nucleotide sequences are shown as sequences 1-12 in a sequence table. The LAMP primer group disclosed by the invention can be used for identifying whether to-be-detected flies belong to three common flies of fresh-eating cherries, i.e., drosophila melanogaster, bactrocera dorsalis and bactrocera tauropean, and can also be used for detecting whether a to-be-detected sample contains three common flies of fresh-eating cherries, i.e., drosophila melanogaster, bactrocera dorsalis and bactrocera tauropean. The method can also be used for distinguishing three common flies of fresh cherries, namely drosophila melanogaster, bactrocera dorsalis and bactrocera cerasus. The LAMP primer group disclosed by the invention has high specificity and high sensitivity, can realize simple, rapid and accurate detection, and is of great significance to the fresh cherry industry and commercial cherry trade.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a LAMP primer set used for identifying common flies that eat fresh cherries and applications thereof, in particular to three common flies that are used to identify fresh cherries - Drosophila melanogaster and Drosophila melanogaster , The LAMP primer set and its application of B. serrata. Background technique [0002] The spotted-winged fruit fly, the fruit fly of orange fruit fly, and the fruit fly of European cherry are common quarantine flies in fresh cherries. Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), also known as Drosophila Suzuki, Drosophila cherry, English name Spotted Wing Drosophila (SWD), is a worldwide pest of great concern, belonging to Diptera, Cyclea, Drosophila, Drosophila, Drosophila subgenus, Drosophila melanogaster species group. The spotted-winged Drosophila has a wide range of hosts, involving more than 60 species of plants from 18 families, incl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6888C12Q1/6844C12Q2600/166C12Q2531/119C12Q2545/113C12Q2565/125Y02A50/30
Inventor 李志红王小雪
Owner CHINA AGRI UNIV
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