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Spiraling whitefly specificity SCAR primer, detecting method and reagent case thereof

A detection kit and the technology of screw whitefly, applied in the field of molecular biology, can solve the problems of no detection method for screw whitefly, and achieve the effects of saving detection time, simple operation process and high detection accuracy

Inactive Publication Date: 2010-04-14
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

But at present, there is no standard detection method for screw whitefly in China

Method used

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  • Spiraling whitefly specificity SCAR primer, detecting method and reagent case thereof
  • Spiraling whitefly specificity SCAR primer, detecting method and reagent case thereof
  • Spiraling whitefly specificity SCAR primer, detecting method and reagent case thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Amplification effect of primer ADZE / ADZF on spiral whitefly

[0035] 1) Preparation of whitefly template DNA

[0036]Place the single-headed whitefly on a parafilm dripped with 20 μL of extraction buffer (50 mmol / L Tris-HCl, 1 mmol / L EDTA, 1% SDS, 20 mmol / L NaCl, pH 8.0), and use the bottom of a 0.2 mL PCR tube as The homogenizer is fully ground, and the homogenate is transferred into a 1.5mL centrifuge tube with a micropipette; then the homogenizer is washed with 100μL buffer solution for 4 times, transferred to the same centrifuge tube, mixed evenly, and 5μL proteinase K (20mg / mL) is added. After mixing thoroughly, place in a water bath at 60°C for 1 hour (mix once in the middle); then bathe in boiling water for 5 minutes, add 220 μL of chloroform / isoamyl alcohol (V:V=24:1) extract, and mix gently for dozens of times. Place on ice for 30 minutes; centrifuge at 4°C and 10,000 r / min for 10 minutes, take the supernatant, add 440 μL pre-cooled absolute ethanol...

Embodiment 2

[0054] Example 2: Amplification effect of primers ADZE / ADZF on different stages and sexes of the spiral whitefly

[0055] 1) Preparation of spiral whitefly template

[0056] Single-headed / single-spirited whiteflies of different stages and sexes were placed on a parafilm dripped with 20 μL of extraction buffer (50 mmol / L Tris-HCl, 1 mmol / L EDTA, 1% SDS, 20 mmol / L NaCl, pH 8.0) , using the bottom of a 0.2mL PCR tube as a homogenizer to fully grind, transfer the homogenate into a 1.5mL centrifuge tube with a micropipette; then wash the homogenizer with 100μL buffer solution for 4 times, transfer to the same centrifuge tube, mix well, add 5 μL of proteinase K (20 mg / mL), mix thoroughly, and place in a water bath at 60°C for 1 hour (mix once in the middle); then in a boiling water bath for 5 minutes, add 220 μL of chloroform / isoamyl alcohol (V:V=24:1) extract , after gently mixing dozens of times, place on ice for 30min; centrifuge at 4°C, 10000r / min for 10min, take the supernatan...

Embodiment 3

[0068] Example 3: Determination of Primers ADZE / ADZF on the Minimum Detection Amount of Spiral Whitefly

[0069] 1) Preparation of spiral whitefly template

[0070] Single-headed spiral whitefly female adults were placed on a parafilm membrane dripped with 20 μL of extraction buffer (50 mmol / L Tris-HCl, 1 mmol / L EDTA, 1% SDS, 20 mmol / L NaCl, pH 8.0), and 0.2 mL of The bottom of the PCR tube was fully ground as a homogenizer, and the homogenate was transferred into a 1.5mL centrifuge tube with a micropipette; then the homogenizer was washed with 100 μL buffer solution four times, transferred to the same centrifuge tube, mixed evenly, and 5 μL of proteinase K (20 mg / mL), after fully mixing, place in a water bath at 60°C for 1 h (mix once in the middle); then bath in boiling water for 5 min, add 220 μL of chloroform / isoamyl alcohol (V:V=24:1) extract, and mix gently for several After ten times, place on ice for 30 minutes; centrifuge at 4°C, 10,000 r / min for 10 minutes, take th...

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Abstract

The invention relates to the field of the molecular biology, in particular to a spiraling whitefly specificity SCAR primer, a detecting method and a reagent case thereof. According to the ribonucleotide sequence of the spiraling whitefly specificity SCAR primer, showing in SEQ- ID- No: 1 and 2, the invention designs a pair of specificity primers according to the unique genome DNA sequence of spiraling whitefly, and the primers only have the proliferation ability for spiraling whitefly, which is a supplement and improvement for spiraling whitefly RAPD technological detecting method.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular, the invention relates to a spiral whitefly-specific SCAR primer, a detection method and a kit. Background technique [0002] The spiral whitefly Aleurodicus dispersus Russell belongs to the order Homoptera, the family Whitefly, the subfamily Whitefly, and the genus Whitefly. It is an invasive pest that seriously endangers agricultural and forestry production. It is listed as A2 quarantine by the Caribbean Regional Plant Protection Organization sexual pests. Spiral whitefly harms 481 species of plants in 90 families, 295 genera, including vegetables, fruit trees, ornamental plants, street trees and forests, among which guava, mulberry, terminal nut, papaya, custard apple, cherry, pepper, eggplant, wolfberry, Bauhinia, hibiscus, maple, dahlia, rhododendron, jasmine, Christmas red, canna, orangutan grass, and amaranthus were severely damaged. There are three main ways of damage, inc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 张桂芬吴霞万方浩刘万学郭建英刘循
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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