Pair of phenacoccus solenopsis specific SS-COI primers, and rapid PCR detection method and kit

A technology for the detection method of fuso mealybug and its detection method, which is applied in the field of molecular biology, can solve the problems that there is no detection method for fuso mealybug, achieve the effect of meeting plant quarantine and pest monitoring/detection, simple operation process, and strong practicability

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

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Problems solved by technology

But at present, there is no standard detect

Method used

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  • Pair of phenacoccus solenopsis specific SS-COI primers, and rapid PCR detection method and kit
  • Pair of phenacoccus solenopsis specific SS-COI primers, and rapid PCR detection method and kit
  • Pair of phenacoccus solenopsis specific SS-COI primers, and rapid PCR detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Implementation Example 1: Amplification effect of primers PSZTF1 / PSZTR1 on Pyococcus hibiscus

[0028] 1) Preparation of mealybug template DNA

[0029] Place a single-headed mealybug (about 1 / 3 body length) on a parafilm membrane dripped with 20 μL of extraction buffer (50 mM Tris-HCl, lmMEDTA, 1% SDS, 20 mM NaCl, pH8.0), and use 0.2 mL of PCR Grind the bottom of the tube as a homogenizer, transfer the homogenate into a 1.5mL centrifuge tube with a micropipette; then wash the homogenizer and Prafilm membrane with 200 μL buffer solution for 4 times, transfer to the same centrifuge tube, mix well, and add 5 μL proteinase K (20mg / mL), mix thoroughly and place in a water bath at 60°C for 1.5h (mixing twice in the middle); then bathe in boiling water for 8min, add 220μL of chloroform / isoamyl alcohol (V:V=24:1) extract, gently After mixing dozens of times, place it on ice for 30 minutes; centrifuge at 4°C and 12000 r / min for 20 minutes, take about 200 μL of the supernatant a...

Embodiment 2

[0043] Implementation example 2: Amplification effect of primers PSZTF1 / PSZTR1 on different stages and ages of P. hibiscus

[0044] 1) Preparation of the template DNA of Pythococcus hibiscus cotton

[0045] Single-headed / single Pythococcus hibiscus of different stages and ages were placed on a parafilm dripped with 20 μL of extraction buffer (50 mM Tris-HCl, 1 mM EDTA, 1% SDS, 20 mM NaCl, pH 8.0). Use the bottom of a 0.2mL PCR tube as a homogenizer to grind thoroughly, transfer the homogenate into a 1.5mL centrifuge tube with a micropipette; then wash the homogenizer and Prafilm membrane with 200μL buffer solution 4 times, transfer to the same centrifuge tube, and mix well , add 5 μL of proteinase K (20mg / mL), mix thoroughly, and then place in a water bath at 60°C for 1.5 hours (mixing twice in the middle); then add 220 μL of chloroform / isoamyl alcohol (V:V=24:1) in a boiling water bath for 8 minutes After gently mixing the extract for dozens of times, place it on ice for 30 ...

Embodiment 3

[0057] Implementation Example 3: Amplification results of primers PSZTF1 / PSZTR1 on Pythococcus hibiscus cotton collected from 14 different regions in 7 provinces and cities in my country and Pythias spongiosa from Pakistan intercepted by ports

[0058] 1) Preparation of the template DNA of Pythococcus hibiscus cotton

[0059] A single adult female P. solani (about 1 / 3 of the body length) was placed on a parafilm dripped with 20 μL of extraction buffer (50 mM Tris-HCl, 1 mM EDTA, 1% SDS, 20 mM NaCl, pH 8.0). The bottom of the 0.2mL PCR tube 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 Prafilm membrane were washed with 200 μL buffer solution for 4 times, transferred to the same centrifuge tube, and mixed. Add 5 μL of proteinase K (20 mg / mL), mix thoroughly, and place in a 60°C water bath for 1.5 h (mixing twice in the middle); then add 220 μL of chloroform / isoamyl alco...

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Abstract

The invention belongs to the field of molecular biology, and relates to a pair of phenacoccus solenopsis specific SS-COI primers, a rapid PCR detection method, and a kit. The primers, the method and the kit are suitable to be used for detecting phenacoccus solenopsis. According to the invention, according to specific mitochondrial DNA sequence of phenacoccus solenopsis, a pair of specific primers is designed. The primers only have amplification capacities upon phenacoccus solenopsis, wherein an amplification product size is 546bp. The primers also have good detection method upon single egg and new nymph. The pair of primers is a supplementation and improvement to a phenacoccus solenopsis mtDNA COI technical detection method. 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 a form of a kit in our ports, organic vegetable and organic fruit production base, cut flower production base, and transportations of vegetables, ornamental plants, and fruit trees seedlings/plants.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular, a pair of hibiscus cotton specific SS-COI primers, a rapid PCR detection method and a kit of the invention. Background technique [0002] Phenacoccus solenopsis Tinsley, belonging to Hemiptera, Phenacoccus family, and Phenyococcus genus, is a worldwide quarantine pest. The List of Quarantine Pests of Imported Plants of the People's Republic of China, and requires all entry-exit inspection and quarantine agencies to strengthen the inspection and quarantine of host plants in countries or regions where the insects occur. The range of hosts for Pythococcus hibiscus is very wide. Existing studies have shown that there are 154 species of host plants in 53 families, including crops, garden plants, weeds and shrubs, etc. The severely damaged plants include cotton, hibiscus, Abutilon, etc.; tomato, eggplant, pepper, wolfberry, black nightshade, etc. of Solanaceae; cocklebur, bitter gourd, ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 张桂芬田虎王瑞万方浩马骏周忠实
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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