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Pair of tuta absoluta specific SS-COI primers, and rapid PCR detection method and kit

A specific technology for leafminers, applied in the field of molecular biology, can solve the problem of no detection method for tomato leafminers, and achieve the effects of saving detection time, simple operation process, and improving accuracy and sensitivity

Active Publication Date: 2013-06-05
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

However, there is no standard detection method for tomato leafminer in China at present.

Method used

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  • Pair of tuta absoluta specific SS-COI primers, and rapid PCR detection method and kit
  • Pair of tuta absoluta specific SS-COI primers, and rapid PCR detection method and kit
  • Pair of tuta absoluta specific SS-COI primers, and rapid PCR detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Implementation Example 1: Amplification Effect of Primer TAZJCE1 / TAZJCF1 on Tomato Leaf Miner

[0027] 1) Preparation of template DNA for leaf miner pests

[0028]Place single adult leafminer pests in a 1.5mL centrifuge tube, add liquid nitrogen and grind them thoroughly with a grinding rod, and then wash them with 220 μL buffer solution (50mM Tris-HCl, 1mM EDTA, 1%SDS, 20mM NaCl, pH8. 0) Wash the grinding rod twice, mix well, add 5 μL proteinase K (20mg / mL), mix well, and put it in a water bath at 60°C for 1 hour (mix once in the middle); then add 220 μL chloroform / isoamyl in a boiling water bath for 8 minutes Alcohol (V:V=24:1) extract, mixed gently dozens of times, placed on ice for 30min; centrifuged at 4°C, 12000r / min for 20min, took about 400μL of the supernatant and transferred it to another centrifuge tube, added 800μL Pre-cooled absolute ethanol, mix gently, and place at -20°C for 30min after a small amount of flocculent precipitate appears; centrifuge at 4°C,...

Embodiment 2

[0045] Implementation Example 2: Amplification Effect of Primer TAZJCE1 / TAZJCF1 on Tomato Leaf Miner Eggs and Adult Residues

[0046] (1) Preparation of tomato leaf miner template DNA

[0047] Place single eggs and adult remains of tomato leafminer (including single antenna, head, thorax (1 / 2), abdomen (1 / 5), forewing, hindwing, forefoot, middle foot, hindfoot) Put 20μL of extraction buffer (50mM Tris-HCl, 1mM EDTA, 1%SDS, 20mM NaCl, pH8.0) on the parafilm membrane, and use the bottom of 0.2mL PCR tube as a homogenizer to fully grind, and the homogenate is mixed with a micropipette Transfer the pipette into a 1.5mL centrifuge tube; then wash the homogenizer and Prafilm membrane with 200μL buffer for 4 times, transfer to the same centrifuge tube, mix well, add 5μL proteinase K (20mg / mL), mix well and put at 60℃ Water bath for 1.5 hours (mix once in the middle); then boil water bath for 8 minutes, add 220 μL of chloroform / isoamyl alcohol (V:V=24:1) extract, mix gently dozens of...

Embodiment 3

[0059] Implementation Example 3: Primer TAZJCE1 / TAZJCF1 Determination of the Minimum Detection Threshold of Tomato Leaf Miner

[0060] (1) Preparation of tomato leaf miner template DNA

[0061] Place the female adults of the single-headed tomato leafminer in a 1.5mL centrifuge tube, add liquid nitrogen and grind it thoroughly with a grinding rod, and then wash it with 220 μL buffer solution (50mM Tris-HCl, 1mM EDTA, 1%SDS, 20mM NaCl, pH8 .0) Clean the grinding rod twice, mix well, add 5 μL proteinase K (20mg / mL), mix thoroughly, and put in a water bath at 60°C for 1 hour (mix once in the middle); then add 220 μL chloroform / iso Pentanol (V:V=24:1) extract, mixed gently dozens of times, placed on ice for 30min; centrifuged at 4°C, 12000r / min for 20min, took about 400μL of the supernatant and transferred it to another centrifuge tube, added 800 μL of pre-cooled absolute ethanol, mix gently, and place at -20°C for 30min after a small amount of flocculent precipitate appears; cent...

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PUM

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Abstract

The invention belongs to the field of molecular biology, and relates to a pair of tuta absoluta specific SS-COI primers, a rapid PCR detection method, and a kit. According to the invention, according to specific mitochondrial DNA sequence of tuta absoluta, a pair of specific primers is designed. The primers only have amplification capacities upon tuta absoluta, wherein an amplification product size is 256bp. The primers also have good detection method upon single egg and adult residues. The pair of primers is a supplementation and improvement to tuta absoluta RAPD and rDNA ITS, and mtDNA COI technical detection methods. Also, an SS-COI PCR technology is adopted, such that detection accuracy is improved, and detection time is saved. The primers and the method can be popularized in our ports in a form of a kit.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular, a pair of tomato leafminer-specific SS-COI primers, a rapid PCR detection method and a kit of the invention. Background technique [0002] The tomato leaf miner Tuta absoluta (Meyrick), belonging to the Lepidoptera and the family Lepidae, is a worldwide destructive quarantine pest of tomato, and was listed as a European and Mediterranean Plant Protection Organization (European and Mediterranean Plant Protection Organization, One of the quarantine pests of category A1 of EPPO). Tomato leafminer is an oligophagous pest, which is particularly harmful to tomatoes, and can occur in both field and greenhouse; in addition, it can also damage economic crops such as eggplant, potato, green pepper, ginseng fruit, tobacco, black nightshade, silver-leaved sunflower, and pseudo- Non-cultivated Solanaceae plants such as thorny eggplant and soapy nightshade, as well as natural Solanaceae plants ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 张桂芬刘万学郭建洋万方浩张毅波
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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