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LAMP primer group for identifying bactrocera minax and application of LAMP primer group

A technology of Bactrocera citrus and primer set, which is applied in the biological field, can solve the problems of unsuitable grass-roots departments for promotion and application, difficulties in morphological identification, expensive instruments and equipment, etc., and achieve the effects of avoiding positive sample contamination, easy promotion, 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 Bactrocera citrus and 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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  • LAMP primer group for identifying bactrocera minax and application of LAMP primer group
  • LAMP primer group for identifying bactrocera minax and application of LAMP primer group
  • LAMP primer group for identifying bactrocera minax and application of LAMP primer group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1. Acquisition of primer set and kit for identification of B. citrus fruit fly

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

[0077] 1. Taking the DNA barcode sequence of COI gene in the mitochondrial genome of B. citrus as the target gene, we designed and synthesized primer sets BM1 and BM2 for identification of B. citrus. 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 are B. citrus fruit flies numbered 3 in Table 1, B. citrus fruit flies numbered 6, and B. citrus fruit flies numbered 18.

[0083] 3. Using the genomic DNA of the fruit fly sample as a template, use primer set BM1 or primer set BM2 to perform loop-mediated isothermal amplification to obtain the amplification pr...

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 of citrus fruit fly

[0093] 1. Identify whether the fruit fly to be tested is or is a candidate for B. citrus fruit fly by agarose gel electrophoresis

[0094] 1. Use a blood / cell / tissue sample 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 BM1 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 DNA template, 9 μL primer mix and 2.5 μL sterile ultrapure water. The primer mixture is the mixture composed of each primer in the primer set BM1. In the reaction system, the final concentration of outer primer F3...

Embodiment 3

[0116] Example 3. Specificity experiment

[0117] The fruit fly 1 to be tested is the citrus fruit fly numbered 5.

[0118] The fruit fly 2 to be tested is the citrus fruit fly numbered 6.

[0119] The fruit fly 3 to be tested is the citrus fruit fly numbered 7.

[0120] The fruit fly 4 to be tested is the citrus fruit fly numbered 8.

[0121] The fruit fly 5 to be tested is the citrus fruit fly numbered 9.

[0122] The fruit fly 6 to be tested is the citrus fruit fly numbered 10.

[0123] The fruit fly 7 to be tested is the citrus fruit fly numbered 11.

[0124] The fruit fly 8 to be tested is the citrus fruit fly numbered 12.

[0125] The fruit fly 9 to be tested is the citrus fruit fly numbered 13.

[0126] The fruit fly 10 to be tested is the citrus fruit fly numbered 14.

[0127] The fruit fly 11 to be tested is the citrus fruit fly numbered 15.

[0128] The fruit fly 12 to be tested is the citrus fruit fly numbered 16.

[0129] The fruit fly 13 to be tested is t...

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) primer group for identifying bactrocera minax and application of the LAMP primer group. The LAMP primer group is composed of a prime-F3, a prime-B3, a primer-FIP and a primer-BIP, and nucleotide sequences of the LAMP primer group are sequentially shown as a sequence 1 to a sequence 4. The LAMP primer group provided by the invention can be used for identifying whether to-be-detected fruit flies are or are candidates of citrus fruit flies, and can also be used for detecting whether to-be-detected samples contain or are candidates of citrus fruit flies. The LAMP primer group is adopted for identifying the bactrocera minax, high specificity and high sensitivity are achieved, and simple, convenient, rapid and accurate detection can be achieved. The method has great application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a LAMP primer set for identifying B. citrus fruit fly and its application. Background technique [0002] The citrus fruit fly, Bactrocera minax (Enderlein), belongs to the Tetradacus genus Bactrocera subgenus Tetradacus in the family Tephritidae of the order Diptera. It is one of the pests that seriously endanger citrus. With the increase of domestic citrus planting area and the increasing frequency of citrus fruit trade, the occurrence range and harm of citrus fruit fly are expanding. [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 vertically and horizontally between the sac flaps to eat. The number of larvae that lay eggs and hatch in the early stage is large, which cau...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6888C12Q1/6844C12Q2600/166C12Q2531/119C12Q2565/125C12Q2563/103Y02A50/30
Inventor 李志红杨文钊
Owner CHINA AGRI UNIV
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