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Real-time fluorescence quantification PCR (Polymerase Chain Reaction) method for detecting ustilago scitaminea

A technology of real-time fluorescence quantification and sugarcane smut, applied in fluorescence/phosphorescence, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of high cost, long cycle, unfavorable detection, judgment and control, etc. Achieve the effect of simple operation, high sensitivity and strong specificity

Inactive Publication Date: 2013-07-17
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above-mentioned traditional methods are relatively intuitive, due to the latent infection of the pathogen, the judgment method based on phenotypic symptoms cannot completely determine whether sugarcane is infected by smut, and it is also difficult to judge the amount of spores in it. There are problems of high cost, long period (need to investigate and collect during the occurrence and epidemic period of the whole crop season, and the planting time takes 8-10 months), and are greatly affected by environmental and human factors. However, the method of capturing pathogenic spores The method is also easily affected by weather such as wind direction, air volume operation, and the size and uniformity of sugarcane plants. Bad weather such as rain will further affect the capture of pathogenic spores
In addition, the traditional methods of isolation, purification, cultivation and morphological identification of pathogens usually take a week, which is not conducive to timely detection, judgment and control

Method used

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  • Real-time fluorescence quantification PCR (Polymerase Chain Reaction) method for detecting ustilago scitaminea
  • Real-time fluorescence quantification PCR (Polymerase Chain Reaction) method for detecting ustilago scitaminea
  • Real-time fluorescence quantification PCR (Polymerase Chain Reaction) method for detecting ustilago scitaminea

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Experimental program
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Effect test

Embodiment 1

[0031]Embodiment 1, a kind of real-time fluorescent quantitative PCR method that detects sugarcane smut

[0032] 1. Extraction of Sugarcane Genomic DNA

[0033] Genomic DNA was extracted by the CTAB method, and the specific operations were as follows:

[0034] (1) After wiping the 7 sugarcane samples to be tested with alcohol with a volume concentration of 75%, put them in a mortar, grind them into fine powder with liquid nitrogen, and distribute them in 2 mL centrifuge tubes. The sample volume of each centrifuge tube is 0.2 g;

[0035] (2) Add 900 μL of 2×CTAB extract preheated at 65 °C, shake and mix, incubate in a water bath at 65 °C for 60 min, and mix by inverting every 5 to 10 min;

[0036] (3) After the water bath, take out the centrifuge tube and centrifuge at 12,000 rpm / min for 5 min. Pipette 750 μL of the supernatant into a new 2 mL centrifuge tube, add an equal volume of phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1), shake, 12,000 rpm / min, and...

Embodiment 2

[0066] Embodiment two, the detection of real-time fluorescence quantitative PCR sensitivity

[0067] (1) Comparison test of positive plasmid detection limit

[0068] 100 ng / μL of recombinant b Plasmid DNA was serially diluted 10 times, according to the formula "MW=number of bases×660 dalton / bp, copies / mL=6.02×10 23 ×(concentration g / mL) / (MW g / mol)”, the concentration is 100 ×10 -3 ~100×10 -11 ng / μL diluted sample converted to known copy number is 1.987×10 8 ~1.987×10 1 copies / µL of 8 10-fold gradients b Plasmid DNA template, and then perform real-time fluorescent quantitative PCR and common PCR detection respectively. Among them, the total reaction system of real-time fluorescent quantitative PCR is 25 μL, including 12.5 μL of 2×TaqMan Universal Master Mix, 1 μL of 10 μmol / L quantitative PCR detection primers bEQ-F and bEQ-R, 10 μmol / L Taqman probe 0.2 μL, b Plasmid DNA template 1 μL, ddH 2 Make up to 25 μL with O; the amplification program is: 50 °C, 2 min, 95 °...

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Abstract

The invention relates to a real-time fluorescence quantification PCR (Polymerase Chain Reaction) method for detecting ustilago scitaminea. The method comprises the steps of sugarcane genome DNA (Deoxyribonucleic Acid) extraction, real-time fluorescence quantification PCR detection system establishment, experiment effectiveness judgment and real-time fluorescence quantification PCR detection. According to the real-time fluorescence quantification PCR method, a specificity quantification PCR detection primer and a Taqman probe are designed according to a bE gene of the ustilago scitaminea; with the adoption of the real-time fluorescence quantification PCR method, the quantity of the ustilago scitaminea in a sample to be detected is calculated by a constructed standard curve equation; and the method for detecting the ustilago scitaminea is strong in specificity, high in sensitivity, easy and simple to operate, quick and efficient, and is applicable to resistance identification of the ustilago scitaminea, and to early detection and diagnosis of the ustilago scitaminea infection of farm sugarcane.

Description

technical field [0001] The invention relates to a method for detecting sugarcane pathogenic bacteria, in particular to a real-time fluorescent quantitative PCR method for detecting sugarcane smut, and belongs to the field of biotechnology. Background technique [0002] Sugarcane smut, also known as sugarcane whip smut, smut, the most obvious feature of this disease is a black whip growing from the tip of the diseased plant. Sugarcane smut is caused by the sugarcane smut ( Sporisorium scitamineum ) is an airborne fungal disease caused by the fungus Basidiomycetes Ustilago smut. Seed sugarcane infected with smut germinates early, with slender, light green leaves, small stems, and increased tillers, after which black spikes also grow on the tillers. Sugarcane smut and the fungus-carrying soil are the primary infection sources of sugarcane smut. Chlamydospores are spread by airflow, rain, irrigation water and insects, and then land on the sugarcane buds and hide between the s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12Q1/04G01N21/64
Inventor 苏亚春许莉萍阙友雄薛扳佟郭晋隆林庆良
Owner FUJIAN AGRI & FORESTRY UNIV