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Method for identifying bactrocera minax and primer group used by method

A technology of citrus fruit fly and primer set, which is applied in the biological field, can solve the problems of unsuitable grass-roots departments for popularization and application, difficulty in morphological identification, expensive instruments and equipment, etc., and achieves the effects of avoiding positive contamination of samples, easy popularization and high sensitivity

Pending Publication Date: 2022-08-05
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The morphology of Bactrocera citrus and Bactrocera citrus is similar, and the identification of larvae lacks obvious identification features. To distinguish and identify adults of B. The identification of only chest mane as a distinguishing feature requires professional morphological knowledge reserves, and morphological identification is 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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  • Method for identifying bactrocera minax and primer group used by method
  • Method for identifying bactrocera minax and primer group used by method
  • Method for identifying bactrocera minax and primer group used by method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1. Acquisition of primer sets and kits for identification of B. tangerines

[0076] 1. Screening of primer sets for the identification of B. citrus

[0077] 1. Using the DNA barcode sequence of COI gene in the mitochondrial genome of B. serrata as the target gene, the primer set BT1, BT2, BT3 and BT4 for identification of B. serrata were designed and synthesized. The primer sequences of each primer set are shown in Table 2.

[0078] Table 2

[0079]

[0080]

[0081] 2. Extract the genomic DNA of the fruit fly sample by using a blood / cell / tissue genomic DNA extraction kit to obtain the genomic DNA of the fruit fly sample.

[0082] The fruit fly samples were B. citrus fruit flies numbered 3 in Table 1, B. citrus fruit flies numbered 8, and fruit flies numbered 18.

[0083] 3. Using the genomic DNA of the fruit fly sample as a template, use primer set BT1, primer set BT2, primer set BT3 or primer set BT4 to perform loop-mediated isothermal amplification t...

Embodiment 2

[0092] Example 2. Application of the kit prepared in Example 1 in identifying whether the fruit fly to be tested is or is a candidate for fruit fly

[0093] 1. Identify whether the fruit fly to be tested is or is a candidate for fruit fly

[0094] 1. Use a blood / cell / tissue genomic DNA extraction kit to extract the genomic DNA of the fruit flies to be tested to obtain the genomic DNA of the fruit flies to be tested.

[0095] 2. Using the genomic DNA of the fruit fly to be tested as a template, using the primer set BT1 to perform loop-mediated isothermal amplification to obtain the amplification product.

[0096] 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 the mixture composed of each primer in the primer set BT1. In the reaction system, the final concentration of outer primer F3 and outer primer B3 were both 0.2 μM, and the final concentration of inner primer F...

Embodiment 3

[0116] Example 3. Specificity experiment

[0117] The fruit fly 1 to be tested is the fruit fly numbered as 1.

[0118] The fruit fly 2 to be tested is the fruit fly numbered 2.

[0119] The fruit fly 3 to be tested is the fruit fly numbered 3.

[0120] The fruit fly 4 to be tested is the fruit fly numbered 4.

[0121] The fruit fly 5 to be tested is the citrus fruit fly numbered 5.

[0122] The fruit fly 6 to be tested is the citrus fruit fly numbered 6.

[0123] The fruit fly 7 to be tested is the citrus fruit fly numbered 7.

[0124] The fruit fly 8 to be tested is the citrus fruit fly numbered 8.

[0125] The fruit fly 9 to be tested is the citrus fruit fly numbered 9.

[0126] The fruit fly 10 to be tested is the citrus fruit fly numbered 10.

[0127] The fruit fly 11 to be tested is the citrus fruit fly numbered 11.

[0128] The fruit fly 12 to be tested is the citrus fruit fly numbered 12.

[0129] The fruit fly 13 to be tested is the citrus fruit fly numbered ...

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Abstract

The invention discloses a method for identifying bactrocera minax and a primer group used by the method. The primer group is composed of a prime-F3, a prime-B3, a primer-FIP and a primer-BIP, and nucleotide sequences of the primer group are sequentially shown as a sequence 1 to a sequence 4. The loop-mediated isothermal amplification technology for identifying the bactrocera minax can realize rapid identification of eggs, larvae / adult individuals of the bactrocera minax, has the advantages of rapidness, high efficiency, strong specificity, high sensitivity, low cost, simplicity and convenience in operation and no need of special instruments, is suitable for the field, is easy to popularize in the basic level, and has wide application prospects. A reliable technical basis can be provided for prevention and control of the citrus fruit flies, and the method has important significance on plant quarantine and citrus fruit trade.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for identifying B. tangerines and a primer set used therefor. Background technique [0002] Bactrocera tsuneonis (Miyake), belonging to the Diptera (Diptera) Tephritidae, Bactrocera subgenus Tetradacus, English name Japanese orangefly, is a danger of international concern Pests are also my country's entry and national agricultural phytosanitary pests. In my country, it is mainly distributed in Guangxi Zhuang Autonomous Region, Sichuan Province, Guizhou Province, Yunnan Province, Hunan Province and Taiwan Province, but its distribution is limited, and there are only a few cities and counties in each province. [0003] The citrus fruit fly obligately parasitizes citrus fruit trees, and its larvae feed on the sac flaps in the damaged fruit, and some can also feed on the seeds, and the fruit is flocculent in the later period of damage. The larvae can travel vertical...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6888C12Q1/6844C12Q2531/119C12Q2565/125Y02A50/30
Inventor 李志红杨文钊
Owner CHINA AGRI UNIV
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