One-step dual-specificity SS-COI primer combination of frankliniella occidentalis race and applications thereof

A combination of primers and technology of Western flower thrips, applied in the field of agricultural biology, can solve the problems of high base sequence homology, difficulty in finding specific segments of lupine strains, etc., achieve strong sensitivity, simple operation process, and detection The effect of high accuracy

Active Publication Date: 2019-05-03
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

Although there are about 130 known COI sequences of thrips pests in the current database, there are only 4 known COI sequences for flower thrips pests, and there are only 4 related species, and the western flower thrips greenhouse strain and lupin The homology of base sequences among bean strains is relatively high, and it is difficult to find specific segments targeting the greenhouse strains of Western flower thrips and lupine strains respectively

Method used

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  • One-step dual-specificity SS-COI primer combination of frankliniella occidentalis race and applications thereof
  • One-step dual-specificity SS-COI primer combination of frankliniella occidentalis race and applications thereof
  • One-step dual-specificity SS-COI primer combination of frankliniella occidentalis race and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation of specific combination primers

[0049] The specific segments in the mitochondrial mtDNACOI gene sequence of the western flower thrips green house strain and the lupine strain were respectively used as target sequences, and the strain-specific combined primers (TFZ6 / GRZ32 / LRZ12) were designed, specifically:

[0050] Universal upstream primer TFZ6 for greenhouse and lupine lines:

[0051] 5'-CGACTTAATAACATAAGATTTT-3';

[0052] Greenhouse strain-specific downstream primer GRZ32: 5'-GATGTATCTAAGTCTCGGTCT-3';

[0053] Lupine line-specific downstream primer LRZ12: 5'-GCATAGCATAGATTAGTCCC-3'.

[0054] Using the DNA of the western flower thrips greenhouse strain and the lupine strain as templates, the ratios of primer combinations TFZ6 / GRZ32 / LRZ12 were 1.0 / 0.0 / 1.0, 1.0 / 0.1 / 0.9, 1.0 / 0.2 / 0.8, 1.0 / 0.3 / 0.7, 1.0 / 0.4 / 0.6, 1.0 / 0.5 / 0.5, 1.0 / 0.6 / 0.4, 1.0 / 0.7 / 0.3, 1.0 / 0.8 / 0.2, 1.0 / 0.9 / 0.1, 1.0 / 1.0 / 0.0 μL PCR to determine the best primers Proportions and Reacti...

Embodiment 2

[0056] Embodiment 2 verifies the specificity of combination primer

[0057] Using the combined primer TFZ6 / GRZ32 / LRZ12, using the DNA of the western flower thrips greenhouse strain and the lupine strain as templates, the T. Common large thrips, bean-billed thrips, sweet clover thrips, sucrose-dental thrips, rice simple thrips, ficus broad-tube thrips, ficus female thrips, horizontal thrips and other 14 species A common species of thrips was used as a control for one-step duplex SS-COI PCR amplification.

[0058] Adult thrips insects collected in the field were confirmed by morphological characteristics and DNA barcode identification, DNA was extracted, and stored at -20°C for later use.

[0059] 5 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.

[0060] Such as figure 1 As shown, the target band of 362bp was amplified in the 1 lane...

Embodiment 3

[0061] Embodiment 3 verifies the accuracy rate of combination primer

[0062] Using combined primers TFZ6 / GRZ32 / LRZ12, PCR amplification was carried out using the DNA of T. occidentalis greenhouse strains and lupine strains of different stages and larval instars as templates.

[0063] The eggs, 1-2 instar larvae, prepupa, pupae, male adults and female adults of different developmental stages were collected from single-headed / single-grained western flower thrips greenhouse strains and lupine strains, and DNA was extracted and stored at -20°C for later use.

[0064] The result is as figure 2 As shown, lanes 1 to 7 all show the target band of 362bp greenhouse strain, and lanes 8 to 14 all show the target band of 541bp lupine strain, indicating that the single egg and single egg of the greenhouse strain of western flower thrips and the lupine strain The target gene could be amplified successfully in the first hatchling larvae, 2nd instar larvae, prepupae, pupae, male adults and ...

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Abstract

The invention belongs to the field of agricultural biotechnology, and specifically relates to a one-step dual-specificity SS-COI primer combination of the frankliniella occidentalis race and applications thereof. The one-step dual-specificity SS-COI primer combination of a frankliniella occidentalis race of the invention comprises a universal upstream primer TFZ6 of the greenhouse race and the lupine race, a specific downstream primer GRZ32 of the greenhouse race and a specific downstream primer LRZ12 of the lupine race. The sizes of one-step dual-specificity PCR primer combination amplification products are respectively 362bp and 541bp. The primer combination has good detection effect on single-grain eggs and newly hatched larvae, improves the accuracy of detection of the frankliniella occidentalis greenhouse and the frankliniella occidentalis lupine race, saves detection time, and is suitable for promotion in the form of a kit in imported plant quarantine, domestic plant quarantine and field control.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a one-step dual-specificity SS-COI primer combination and application thereof. Background technique [0002] Western flower thrips (Frankliniella occidentalis Pergande), also known as alfalfa thrips, belongs to Thysanoptera (Thysanoptera) thrips family (Thripidae) flower thrips (Frankliniella), which is native to the northwestern United States. It can spread a variety of crop viruses such as tomato spotted wilt virus, causing the epidemic of viral diseases. Western flower thrips has mixed feeding habits. At present, there are more than 500 species of host plants in more than 60 families. It is a worldwide quarantine pest and an important external and internal quarantine object in my country. There are two strains of Thrips occidentalis, the greenhouse race (GR) and the lupine race (LR), among which the greenhouse race has strong drug resistance. At present,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
Inventor 张桂芬张蓉张毅波田虎冼晓青王玉生刘万学郭建洋万方浩
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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