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Specific COI primer, detection method and reagent kit of Frankliniella occidentalis

A detection kit, a technology for thrips occidentalis, applied in the field of molecular biology, can solve the problems of high cost, time-consuming, difficult to meet high-throughput rapid detection, etc., achieve strong specificity, save detection time, improve accuracy and The effect of sensitivity

Inactive Publication Date: 2010-09-01
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

However, due to the need to sequence the amplification products, the cost is high and time-consuming, so it is difficult to meet the needs of high-throughput rapid detection

Method used

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  • Specific COI primer, detection method and reagent kit of Frankliniella occidentalis
  • Specific COI primer, detection method and reagent kit of Frankliniella occidentalis
  • Specific COI primer, detection method and reagent kit of Frankliniella occidentalis

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Implementation Example 1: Amplification effect of primers FOZLCE / FOZLCF on Thrips occidentalis

[0037] 1) Preparation of thrips template mitochondrial DNA

[0038] Place a single thrips on a parafilm dripped with 20 μL extraction buffer (50 mM Tris-HCl, 1 mM EDTA, 1% SDS, 20 mM NaCl, pH 8.0), and use the bottom of a 0.2 mL PCR tube as a homogenizer to thoroughly grind , the homogenate was transferred into a 1.5mL centrifuge tube with a micropipette; then the homogenizer was washed four times with 200μL buffer, transferred into the same centrifuge tube, mixed evenly, and 5μL proteinase K (20mg / mL) was added, mixed well and then placed in Water bath at 60°C for 1 hour (mix once in the middle); then bath in boiling water for 5 minutes, add 220 μL of chloroform / isoamyl alcohol (V:V=24:1) extract, mix gently dozens of times, and place on ice for 30 minutes; Centrifuge at 4°C and 12000r / min for 15min, take the supernatant, add 440μL of pre-cooled absolute ethanol, mix gentl...

Embodiment 2

[0056] Implementation example 2: Amplification effect of primers FOZLCE / FOZLCF on different stages and sexes of Thrips western flower

[0057] 1) Preparation of template mitochondrial DNA of Thrips occidentalis

[0058] A single head / single Western flower thrips of different stages and sexes was placed on the 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 0.2 mL 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 200 μL buffer solution for 4 times, transferred to the same centrifuge tube, mixed evenly, and 5 μL proteinase K ( 20mg / mL), mix thoroughly and place in a water bath at 60°C for 1h (mix once in the middle); then bathe in boiling water for 5min, add 220μL of chloroform / isoamyl alcohol (V:V=24:1) extract, and mix gently After dozens of times, place on ice for 30 minute...

Embodiment 3

[0070] Implementation Example 3: Primer FOZLCE / FOZLCF Determination of the Minimum Detection Amount of Western Flower Thrips

[0071] 1) Preparation of template mitochondrial DNA of Thrips occidentalis

[0072] Place a single adult female of Thrips occidentalis 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 use the bottom of a 0.2 mL PCR tube as a homogenate Use a micropipette to transfer the homogenate into a 1.5mL centrifuge tube; then wash the homogenizer 4 times with 200μL buffer solution, transfer to the same centrifuge tube, mix well, add 5μL proteinase K (20mg / mL), and mix thoroughly After homogenization, put it 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, mix it gently dozens of times, and put it on ice Place it for 30 minutes; centrifuge at 4°C, 12000r / min for 15min, take ...

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Abstract

The invention relates to the field of molecular biology, in particular to a specific COI primer, a detection method and a reagent kit of Frankliniella occidentalis. The nucleotide sequences of the specific COI primer of the Frankliniella occidentalis of the invention are shown as SEQ IDNo: 1 and 2. According to the specific mitochondrion DNA sequence of the Frankliniella occidentalis, the invention designs a pair of specific primers which only have amplification capability on the Frankliniella occidentalis. The invention is the supplement and improvement on the RFLP technology and the mtDNA COI technology detection method of the Frankliniella occidentalis.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular, the invention relates to specific COI primers, detection methods and kits of Thrips occidentalis. Background technique [0002] Western flower thrips Frankliniella occidentalis (Pergande) belongs to the Thysanoptera, Sawtails, Thripaceae, Flower Thrips genus, is a worldwide quarantine pest, the European and Mediterranean Plant Protection Organization (EPPO) listed it as A2 Quarantine pest, also of quarantine importance to the Caribbean Regional Plant Protection Commission (CPPC) and the International Organization for Plant Protection and Animal Health (OIRSA). Thrips occidentalis is a polyphagous pest with a wide range of hosts. Currently, there are more than 500 species of known host plants in 62 families, and with the deepening of research, new species of host plants are constantly being discovered. The host plants of Thrips occidentalis include various important economic crops,...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/12C12Q1/68
Inventor 闵亮张桂芬孟祥钦刘循万方浩刘万学郭建英周忠实王文凯
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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