Specific SS-COI primer for spodoptera frugiperda and application of specific SS-COI primer

A specific technology of fall armyworm, which is applied in the field of agricultural biology, can solve the problems of difficult to find the specific segment of fall armyworm and high homology, and meet the requirements of plant quarantine and pest monitoring/detection and control , Easy and fast operation, simple operation process

Inactive Publication Date: 2020-06-05
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the analysis shows that the base sequence homology between the above-mentioned different species is relatively high, and it is difficult to find specific segments targeting Spodoptera frugiperda

Method used

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  • Specific SS-COI primer for spodoptera frugiperda and application of specific SS-COI primer
  • Specific SS-COI primer for spodoptera frugiperda and application of specific SS-COI primer
  • Specific SS-COI primer for spodoptera frugiperda and application of specific SS-COI primer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The preparation of embodiment 1 Spodoptera frugiperda specific primer

[0027] Using the specific segment in the mtDNA COI genome sequence of Spodoptera frugiperda as the target sequence, the specific primers (SFZYF3 / SFZYR3) for Spodoptera frugiperda were designed, and its sequence is:

[0028] Primer SFZYF3: 5'-TTAGATTTCGATCAGTAAGTAAT-3';

[0029] Primer SFZYR3: 5'-TTTAACTTTATTAATTTCTAGTAGC-3'.

[0030] Using Spodoptera frugiperda DNA as template, temperature gradient PCR with annealing temperature of 46-53°C was carried out to determine the optimal amplification conditions. For the SFZYF3 / SFZYR3 primer pair, a single band of 323bp can be obtained at the annealing temperature of 52°C and 53°C, and the amplified band is clearer and brighter when the annealing temperature is 53°C, therefore, the annealing temperature is chosen to be 53°C.

Embodiment 2

[0031] The specific amplification effect of embodiment 2 primers to Spodoptera frugiperda

[0032] Using primers SFZYF3 / SFZYR3 to use Spodoptera frugiperda DNA as a template, Spodoptera litura, Spodoptera litura, Spodoptera spp., Spodoptera, yellow cutworm, Helicoverpa armigera, Armyworm orientalis, Prunus chinensis, Asiatic Eleven other common species of moth moths such as corn borer, rice leaf roller, and meadow borer were used as controls for SS-COI PCR amplification.

[0033] The moth moth larvae (2nd or 5th instar) collected in the field were confirmed by morphological characteristics and DNA barcode identification, and DNA was extracted and stored at -20°C for later use.

[0034] 7 μL of the PCR product was separated by electrophoresis on a 1.0% (weight / volume) agarose gel containing Gold view, and then judged by the size of the amplified product in a gel imaging system.

[0035] Such as figure 1 As shown, the target band of 323bp has been amplified in the Spodoptera f...

Embodiment 3

[0036] Example 3 Amplification effect of primers SFZYF3 / SFZYR3 on different stages and larval instars of Spodoptera frugiperda

[0037]Using primers SFZYF3 / SFZYR3, DNA of Spodoptera frugiperda in different stages and different larval instars was used as template for PCR amplification.

[0038] Single head / single Spodoptera frugiperda of different development stages (eggs, 1st-6th instar larvae, female pupae and male pupae, female adult and male adulthood) were taken (the 2nd instar larvae took 1 / 2 body length, and the 3rd instar larvae Take 1 / 4 body length, 1 / 4 abdomen for 4th instar larvae, 1 / 8 abdomen for 5th-6th instar larvae; 1 / 4 abdomen for pupae and adults), extract DNA, store at -20°C for later use.

[0039] The result is as figure 2 As shown, lanes 1 to 11 all show the target band of 323bp, indicating that the single eggs and single-headed larvae, 2nd instar larvae, 3rd instar larvae, 4th instar larvae, 5th instar larvae, and 6th instar larvae of Spodoptera frugiperd...

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Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to a specific SS-COI primer for spodoptera frugiperda and application of the specific SS-COI primer. Thespecific primer only has amplification capacity for the spodoptera frugiperda, the size of an amplified product is 323 bp, and the specific primer has a good detection effect on single-grain eggs andadult residual bodies. The specific primer improves the accuracy of detection of spodoptera frugiperda, saves the detection time and is suitable to be popularized in border trade quarantine, dynamic detection of population dispersion and field control in a kit form.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a Spodoptera frugiperda specific SS-COI primer and an application thereof. Background technique [0002] Spodoptera frugiperda JE Smith, also known as fall armyworm, pseudo armyworm, alfalfa worm, beet armyworm, belongs to Lepidoptera, Noctuidae, Spodoptera genus, native to America, is a world migratory agricultural pests. It has a wide range of hosts, including 350 species of plants in 76 families, especially Gramineae, Compositae and Leguminous plants. The larvae feed on the leaves, eat the tender core, stems and reproductive organs, causing serious damage, and are highly diffusible, easily breaking out into disasters. Therefore, strengthening the detection and monitoring of Spodoptera frugiperda is a necessary prerequisite for ensuring agricultural safety. [0003] The larvae of Spodoptera frugiperda are boring insects, which often occur secretly, espec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/124
Inventor 张桂芬杨安沛郭建洋王玉生张毅波刘万学万方浩王浩毕思言
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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