Nosema pernyi template DNA extraction method and application of nosema pernyi template DNA extraction method to molecular diagnosis

A technology for microsporidia and molecular diagnosis, applied in microorganism-based methods, DNA preparation, recombinant DNA technology, etc., can solve the problems of low detection sensitivity, cumbersome operation, low throughput, etc., and achieve high throughput and interference factors. Less, overcome the effect of tissue blackening

Active Publication Date: 2017-11-28
辽宁省农业科学院大连生物技术研究所
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  • Abstract
  • Description
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Problems solved by technology

[0008] The present invention aims at various problems listed in the background technology such as cumbersome operation, low throughput and low detection sensitivity of the current tussah microsporidia detection technology, and provides a method for obtaining tussah microsporidia template DNA and a kit thereof, and discloses application in molecular diagnostics

Method used

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  • Nosema pernyi template DNA extraction method and application of nosema pernyi template DNA extraction method to molecular diagnosis
  • Nosema pernyi template DNA extraction method and application of nosema pernyi template DNA extraction method to molecular diagnosis
  • Nosema pernyi template DNA extraction method and application of nosema pernyi template DNA extraction method to molecular diagnosis

Examples

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Effect test

Embodiment 1

[0062] Acquisition of template DNA for No. tussah mori and diagnosis of No. tussah by SYBR Green fluorescence quantitative PCR

[0063] Select a diseased pupa infected with No. tussah mori, and put a small amount of tissue into two 1.5mL EP tubes, numbered S-1 and FT-1 respectively. Add 200 μL of lysate S (0.2M NaOH, 1% SDS) to tube S-1, oscillate to mix, let it stand at room temperature for 40 minutes, add magnetic bead suspension 10 μL (sigma) and absolute ethanol 150mL, oscillate gently to mix , place the EP tube on a magnetic stand for separation, and when the magnetic beads are completely separated from the liquid phase, pour off the liquid phase, remove the magnetic stand, add 150 μL of 70% ethanol, oscillate and mix well, place on the magnetic stand for separation, discard For liquid, remove the magnetic stand, add 150 μL of 70% ethanol again, oscillate, mix and place on the magnetic stand for separation, pipette the liquid as much as possible, remove the magnetic stand...

Embodiment 2

[0071] Acquisition of template DNA of No. tussah silkworm and routine PCR diagnosis of No. tussah silkworm.

[0072]Four diseased pupae infected with No. tussah silkworm were selected, numbered S1, S2, S3 and S4 respectively. A small amount of tissue was taken from S1 and S2 respectively. Among them, S1 used a scalpel to cut an incision of about 5 mm on the back of the pupae, quickly picked up a little tissue with a toothpick, and then disinfected the incision with 75% alcohol cotton. Make a wax seal. Put the S1 pupa into the cocoon shell, seal the cocoon shell with adhesive tape, place it at room temperature (15-30°C), and observe the hatching situation. Take a small amount of pupal skin and pupal blood mixture in S3, and take a small amount of pupal skin and tissue mixture in S4, and place them in four 1.5mL EP tubes respectively. Add 200 μL of lysate S (0.2M NaOH, 1% SDS) respectively, oscillate and mix well, leave at room temperature for half an hour, add 10 μL of magnet...

Embodiment 3

[0077] Choose 60 female moths that have laid eggs, numbered respectively 1#-60#. A small amount of tissue was picked and placed in 60 1.5mL EP tubes containing 200μL 0.2M KOH (among them, 4 samples were taken from 2 eggs each due to the small amount of tissue in the belly of the moth). Freeze the tubes at -20°C, remove and thaw after 30 days, transfer 100 μL of the lysate supernatant in each EP tube to different sample wells in a 96-well plate, add 10 μL of magnetic bead suspension (Sigma) and 75 mL of absolute ethanol , gently oscillate and mix, put the EP tube on a 96-well magnetic stand for separation, wait until the magnetic beads are completely separated from the liquid phase, pour off the liquid phase, remove the magnetic stand, add 100 μL of 70% ethanol each, oscillate and mix Then put it on the magnetic stand for separation, discard the liquid, remove the magnetic stand, add 100 μL of 70% ethanol again, oscillate and mix well, and place it on the magnetic stand for sep...

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Abstract

The invention discloses a rapid nosema pernyi template DNA extraction method and application thereof. The rapid nosema pernyi template DNA extraction method specifically comprises the step of releasing, enrichment, separation and purification of DNA through alkaline lysis and magnetic beads. According to the method, operation is simple and rapid, the cost is low, the flux is high, living pupa sampling can be achieved, the antijamming capability is high, the contamination risk is low, the quality of extracted DNA is high, automatic extraction can be achieved, and the method can be widely applied to molecular diagnosis; the detection flux and the operation convenience can be remarkably improved, the false positive rate of detection can be obviously decreased, and the detection sensitivity and accuracy and the detection efficiency are improved; and through an automatic series nucleic acid extraction instrument, a PCR and a preferred result-viewable PCR technique, a visual and automatic nosema pernyi analysis technique which can integrate batched DNA extraction on a porous plate and molecular diagnosis can be achieved. The rapid nosema pernyi template DNA extraction method and the application thereof have important significance for centralized quarantine of antheraea pernyi granulosis and conservation of genetic resources in the antheraea pernyi industry.

Description

technical field [0001] The invention belongs to the technical field of DNA extraction method and application of pathogenic microorganisms, and in particular relates to a method for rapidly obtaining template DNA of tussah mori microsporidia and its application in molecular diagnosis. Background technique [0002] The tussah silkworm (Antheraea pernyi) originated in China and is one of the most valuable biological resources for human beings. my country is the largest country in the world's tussah industry, and its cocoon production accounts for 90% of the world's total cocoon production. At present, the annual output of tussah silkworm cocoons in my country is more than 80,000 tons, with a direct output value of 2.5 billion yuan, and an output value of more than 18 billion yuan in cocoon processing and comprehensive utilization. The tussah industry has become a dominant industry that cannot be replaced in mountainous rural areas of our country. However, since the tussah ind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68C12Q1/04C12R1/90
CPCC12N15/1013C12Q1/6806C12Q1/6893C12Q2523/308
Inventor 李佩佩范琦米锐岳冬梅赵振军叶博王林美张波
Owner 辽宁省农业科学院大连生物技术研究所
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