A pair of Paraleyrodes pseudonaranjae Martin specific SS-COI primers, rapid PCR detection method and kit

A detection kit and detection method technology, applied in the field of molecular biology, can solve the problems of reduced product yield and quality, large insect population density, deformity, etc., and achieve the effects of improving accuracy and sensitivity, simple operation process, and saving detection time

Active Publication Date: 2013-03-06
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

One is direct harm, that is, adults and nymphs prick and suck host plant juice on the back and front of leaves. Due to their strong reproductive ability, fast development speed, and large population density, the growth and development of host plants are seriously hindered, and leaves turn yellow. The second is indirect damage, that is, adults and nymphs secrete a large amount of wax powder, wax silk and honeydew on the back of the host plant leaves, making the back of the leaves appear snow-white, and at the same time induce serious coalescence. Tobacco disease, the leaf surface is covered with a layer of black mold, which seriously affects the photosynthesis, respiration and transpiration of the host plant leaves, reducing the yield and quality of the p...

Method used

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  • A pair of Paraleyrodes pseudonaranjae Martin specific SS-COI primers, rapid PCR detection method and kit
  • A pair of Paraleyrodes pseudonaranjae Martin specific SS-COI primers, rapid PCR detection method and kit
  • A pair of Paraleyrodes pseudonaranjae Martin specific SS-COI primers, rapid PCR detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Amplification effect of primers PPZYF / PPZYR on whitefly

[0029] 1) Preparation of whitefly template DNA

[0030]A single whitefly was placed on a parafilm membrane with 20 μL of extraction buffer (50 mM Tris-HCl, 1 mM EDTA, 1% SDS, 20 mM NaCl, pH 8.0), and the bottom of a 0.2 mL PCR tube was used as a homogenizer to fully grind it. , the homogenate was transferred into a 1.5mL centrifuge tube with a micropipette; then the homogenizer was washed twice with 200μL buffer, transferred to the same centrifuge tube, mixed, and 5μL proteinase K (20mg / mL) was added. 60°C water bath for 1 hour (mixing once in the middle); then boiling water bath for 8 minutes, add 220 μL of chloroform / isoamyl alcohol (V:V=24:1) extract, mix gently for dozens of times, and place on ice for 30 minutes ; 4°C, 12000r / min centrifugation for 20min, take the supernatant, add 440μL of pre-cooled absolute ethanol, mix gently, and place at -20°C for 30min after a small amount of flocculent pre...

Embodiment 2

[0047] Example 2: Amplification effect of primers PPZYF / PPZYR on different insect states and sexes

[0048] 1) Preparation of template DNA of double hooked whitefly

[0049] Single-headed / single-grained whiteflies of different worm states and sexes were placed on a parafilm membrane with 20 μL of extraction buffer (50 mM Tris-HCl, 1 mM EDTA, 1% SDS, 20 mM NaCl, pH 8.0), and 0.2 mL The bottom of the PCR tube was fully ground as a homogenizer, and the homogenate was transferred into a 1.5 mL centrifuge tube with a micropipette; then the homogenizer was washed twice with 200 μL buffer, transferred to the same centrifuge tube, mixed, and 5 μL proteinase K ( 20mg / mL), fully mixed and then placed in a 60°C water bath for 1 hour (mixing once in the middle); then a boiling water bath for 8 minutes, adding 220 μL of chloroform / isoamyl alcohol (V:V=24:1) extract, and mixing gently After tens of times, place on ice for 30min; centrifuge at 4°C, 12000r / min for 20min, take the supernatant...

Embodiment 3

[0061] Embodiment 3: Determination of primer PPZYF / PPZYR on detection threshold of whitefly

[0062] 1) Preparation of template DNA of double hooked whitefly

[0063] Single-headed whitefly female adults were placed on a parafilm membrane dripped with 20 μL of extraction buffer (50 mM Tris-HCl, 1 mM EDTA, 1% SDS, 20 mM NaCl, pH 8.0), and the bottom of a 0.2 mL PCR tube was used as a homogenate. The homogenizer was fully ground, and the homogenate was transferred into a 1.5 mL centrifuge tube with a micropipette; then the homogenizer was washed twice with 200 μL buffer, transferred to the same centrifuge tube, mixed, and 5 μL proteinase K (20 mg / mL) was added. After homogenization, place in a water bath at 60°C for 1 h (mixing once in the middle); then in a boiling water bath for 8 min, add 220 μL of chloroform / isoamyl alcohol (V:V=24:1) extract, mix gently for dozens of times, then freeze Place on the top for 30min; 4°C, 12000r / min centrifugation for 20min, take the supernata...

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Abstract

The invention relates to the field of molecular biology, and in particular relates to a pair of Paraleyrodes pseudonaranjae Martin specific SS-COI primers, a rapid PCR detection method and a kit. The Paraleyrodes pseudonaranjae Martin specific SS-COI primers comprise a primer PPZYF:5'-TAAAGGAACTAATCAATTTCCAAATCCC-3', and a primer PPZYR:5'-GGTCAACAAATCATAAAGATATTGGGT-3'. The primers PPZYF and PPZYR designed according to specific SS-COI mark of Paraleyrodes pseudonaranjae Martin can amplify segments of 233bp in the rapid PCR detection of Paraleyrodes pseudonaranjae Martin, and can not only detect single-head adult, two-age, three-age, and four-age nymph but also detect single spawn and single head newly-hatched nymph.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to a pair of SS-COI primers specific to the whitefly, a rapid PCR detection method and kit of the present invention. Background technique [0002] Double hooked whitefly Paraleyrodes pseudonaranjae Martin, belonging to the order Homoptera, whitefly family, and the genus of whitefly, is a worldwide quarantine pest. There are many plant hosts of the whitefly, including 30 families, 49 genera and 63 species, including 9 species in Rutaceae, 6 species in Moraceae, 5 species in Leguminosae and Rubiaceae, 4 species in Euphorbiaceae, and 3 species in Annuaceae , Palmaceae, Lauraceae, Myrtleaceae, Garcinia, Rhododendron, Sapinaceae and Sapinaceae, 2 species each, Traveler Bananaaceae, Butterflyaceae, Anacardiaceae, Compositae, Sanbai grass 1 species each. Among them, 7 families, including Lauraceae, Strelitziaceae, Combretaceae, Bombacaceae, Compositae, Magnoliaceae, and Elaeocarp...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 张桂芬万方浩郭建洋郭建英刘万学曹凤勤
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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