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Phenacoccus manihoti matile-ferrero specificity SS-COI primer pair as well as method and kit for quick PCR detection

A cassava mealybug and detection kit technology, applied in the field of molecular biology, to achieve the effects of improving accuracy and sensitivity, high application value, and saving detection time

Active Publication Date: 2015-01-21
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, at present, there is no standard detection method for cassava mealybug in China, and the industry standard "Quarantine and Identification Method of Cassava mealybug" based on morphological characteristics has not yet been included in the formulation plan

Method used

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  • Phenacoccus manihoti matile-ferrero specificity SS-COI primer pair as well as method and kit for quick PCR detection
  • Phenacoccus manihoti matile-ferrero specificity SS-COI primer pair as well as method and kit for quick PCR detection
  • Phenacoccus manihoti matile-ferrero specificity SS-COI primer pair as well as method and kit for quick PCR detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028]Example 1: Amplification effect of primers PMZWMEF1 / PMZWMER1 on cassava mealybug

[0029] 1) Preparation of mealybug template DNA

[0030] Place the single-headed mealybug on the parafilm membrane dripped with 20 μL extraction buffer (10 mM Tris-HCl, 50 mM EDTA, 0.5% SDS, 100 mM NaCl, 0.2% β-mercaptoethanol, pH 8.0), and use a 0.2 mL PCR tube The bottom was used as a homogenizer to grind thoroughly, and the homogenate was transferred into a 1.5mL centrifuge tube with a micropipette; then the homogenizer and Parafilm membrane were washed twice with 180 μL buffer solution, transferred to the same centrifuge tube, mixed evenly, and 5 μL proteinase K ( 20mg / mL), mix thoroughly and place in a water bath at 65°C for 40 minutes (mixed twice in the middle); then bath in boiling water for 8 minutes, add 200 μL of chloroform / isoamyl alcohol (V:V=24:1) extract, and mix gently After dozens of times, place it on ice for 30 minutes; centrifuge at 4°C and 7500r / min for 10 minutes, tak...

Embodiment 2

[0044] Example 2: Amplification effect of primers PMZWMEF1 / PMZWMER1 on cassava mealybugs of different stages, ages, residues and host plants

[0045] 1) Preparation of cassava mealybug template DNA

[0046] Single / single-grained cassava mealybugs of different stages (adult, egg, nymph) and instars (1st, 2nd, 3rd instar) from pumpkin, and single female adults (from pumpkin and cassava, respectively) and its head, thorax, abdomen, and feet (all from pumpkin) were placed in drops of 20 μL extraction buffer (10 mM Tris-HCl, 50 mM EDTA, 0.5% SDS, 100 mM NaCl, 0.2% β-mercaptoethanol, pH 8.0) Use the bottom of a 0.2mL PCR tube as a homogenizer to grind fully, and transfer the homogenate into a 1.5mL centrifuge tube with a micropipette; then wash the homogenizer and Parafilm membrane twice with 180μL buffer, and transfer it to the same Centrifuge the tube, mix well, add 5 μL proteinase K (20mg / mL), mix well, and put it in a water bath at 65°C for 40 minutes (mixing twice in the middl...

Embodiment 3

[0058] Embodiment 3: Primer PMZWMEF1 / PMZWMER1 is to the determination of cassava mealybug detection threshold

[0059] 1) Preparation of cassava mealybug template DNA

[0060] A single adult female of P. sockensis was placed on a parafilm membrane dripped with 20 μL of extraction buffer (10 mM Tris-HCl, 50 mM EDTA, 0.5% SDS, 100 mM NaCl, 0.2% β-mercaptoethanol, pH 8.0) at 0.2 The bottom of the PCR tube was used as a homogenizer to fully grind, and the homogenate was transferred into a 1.5mL centrifuge tube with a micropipette; then the homogenizer and the Parafilm membrane were washed twice with 180 μL buffer solution, transferred to the same centrifuge tube, mixed evenly, and added 5 μL of proteinase K (20mg / mL), mix thoroughly and place in a water bath at 65°C for 40 minutes (mixed twice in the middle); then in a boiling water bath for 8 minutes, add 200 μL of chloroform / isoamyl alcohol (V:V=24:1) extract , after gently mixing dozens of times, place on ice for 30min; centri...

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Abstract

The invention relates to a phenacoccus manihoti matile-ferrero specificity SS-COI primer pair, as well as a method and kit for quick PCR detection. The primer pair comprises primer PMZWMEF1:5'-CTTGATAAAACAGGAATTGAG-3' and primer PMZWMER1:5'-CCTTTGATGATTTCTTCTTCT-3'. The primer has amplification capacity just to the phenacoccus manihoti matile-ferrero, has favorable detection effect on eggs at the beginning of nympha and imago residue, and is supplement and modification for morphological detection identification method for the phenacoccus manihoti matile-ferrero; moreover, the method adopts the SS-COI PCR technology, improves accuracy of the detection, saves detection time, and can be popularized in a kit form in ports, cassava production bases, organic vegetable and fruit production bases, fresh and cut flower production bases, cotton planting zones, and transportation of vegetables, adornment plants, fruit germchits / plants.

Description

technical field [0001] The invention relates to the field of molecular biology. Specifically, the invention relates to a cassava mealybug specific SS-COI primer pair, a rapid PCR detection method and a kit. Background technique [0002] Cassava mealybug (Cassava mealybug) Phenacoccus manihoti Matile-Ferrero, belongs to Hemiptera, Mealyacidae, Mealybug genus, is a worldwide quarantine pest, on June 20, 2011 the Ministry of Agriculture of the People's Republic of China and the State Quality The General Administration of Supervision, Inspection and Quarantine jointly issued an announcement to include it in the "List of Imported Plant Quarantine Pests of the People's Republic of China" (Announcement No. Inspection and quarantine of host plants in countries or regions where pests occur. Cassava mealybug eats cassava, especially sweet cassava, and often breaks out on cassava, but there is no report on its host plant so far. Existing studies have shown that the host plants of cas...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888
Inventor 张桂芬王玉生田虎万方浩王瑞冼晓青
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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