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Polymerase chain reaction (PCR) detection kit for maize curvularia leaf spot and detection method for PCR detection kit

A Curvularia leaf spot fungus and detection kit technology, applied in the field of fungal disease detection, can solve the problems of time-consuming, unable to monitor and control the spread of pathogenic bacteria in time, and achieve the effect of high sensitivity and high accuracy

Active Publication Date: 2013-08-21
贵州益百亿生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] An object of the present invention is to provide a PCR detection kit for Curvularia maize leaf spot to be used to solve the existing problems that the detection and identification methods of Curvularia maize leaf spot are time-consuming, unable to monitor and control the spread of pathogenic bacteria in time; the present invention Another purpose of is to provide the PCR detection method of Curvularia maize leaf spot

Method used

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  • Polymerase chain reaction (PCR) detection kit for maize curvularia leaf spot and detection method for PCR detection kit
  • Polymerase chain reaction (PCR) detection kit for maize curvularia leaf spot and detection method for PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, detection Curvularia maize leaf spot mycelium

[0048] 1) Detection kit

[0049] Design three specific primers of Curvularia zea Leaf Spot as follows to form two pairs of primers:

[0050] The first pair: upstream primer 5`-ACGGAGGATGCGGTATGG-3` (SEQ ID NO: 1)

[0051] Downstream primer 5`-CCTCGGAATCGTGCTTTT-3` (SEQ ID NO: 3)

[0052] Second pair: upstream primer 5`-GGGGCTCGCAGGCTAATGT-3` (SEQ ID NO: 2)

[0053] Downstream primer 5`-CCTCGGAATCGTGCTTTT-3` (SEQ ID NO: 3)

[0054] The above sequence was synthesized using a DNA synthesizer.

[0055] Prepare 2 sets of PCR reaction systems in the kit: the first set of reaction system consists of the following components: 1ml solution, 100μl 10×PCR reaction buffer, 20μl dNTP with a concentration of 10mM, 200 units of Taq enzyme, with a concentration of 10mM 10mM 40 μl each of the first pair of upstream and downstream primers, and the rest is ultrapure water; the second set of reaction system consists ...

Embodiment 2

[0062] Embodiment 2, detect the curvularia leaf spot bacterium in the leaf spot type leaf of corn

[0063] 1) Detection kit: use the same reaction system as in Example 1.

[0064] 2) Detection method:

[0065] One, extract pathogenic DNA from the maize leaf that produces leaf spot:

[0066] Rinse the diseased corn leaves with distilled water, absorb the water with absorbent paper; grind the leaves with liquid nitrogen and quickly transfer them to a 1.5mL centrifuge tube; add 600 μL of CTAB extract preheated for 30 minutes (65°C), and place in a 65°C water bath 30 min, shake once every 10 min, then add 500 μL chloroform:isoamyl alcohol (24:1) to the centrifuge tube, lightly pump for 10 min, centrifuge at 12000 rpm for 10 min at 4°C; take the supernatant, and then add the supernatant With the same volume of chloroform: isoamyl alcohol (24:1), lightly pump for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C; take the supernatant; add 2 / 3 volume of pre-cooled isopro...

Embodiment 3

[0068] Embodiment 3, detection Curvularia leaf spot bacteria in necrotic corn leaves

[0069] 1) Detection kit: use the same reaction system as in Example 1.

[0070] 2) Detection method:

[0071] One, extract pathogenic DNA from the corn necrosis type leaf:

[0072] Rinse the necrotic corn leaves with distilled water, absorb the water with absorbent paper; grind the leaves with liquid nitrogen and quickly transfer them to a 1.5mL centrifuge tube; add 600 μL of CTAB extract preheated for 30 minutes (65°C), Bath in water for 30 min, shake once every 10 min, then add 500 μL chloroform: isoamyl alcohol (24:1) to the centrifuge tube, pump lightly for 10 min, centrifuge at 12000 rpm for 10 min at 4 °C; take the supernatant, and then add the supernatant Chloroform with the same volume of liquid: isoamyl alcohol (24:1), pump lightly for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C; take the supernatant; add 2 / 3 volume of pre-cooled 90% ethanol to the supernatant, an...

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Abstract

The present invention relates to a PCR detection kit and a detection method for Curvularia maize leaf spot bacteria. The PCR detection kit for Curvularia maize leaf spot bacteria comprises 2 sets of PCR reaction systems, the first set of reaction systems comprising two specific primers SEQIDNO The first pair of primers consisting of :1 and SEQIDNO:3, the second set of reaction system includes the second pair of primers consisting of two specific primers SEQIDNO:2 and SEQIDNO:3. The present invention designs specific amplification primers according to the clg2p gene of Curvularia maize leaf spot, and constructs a PCR detection kit for Curvularia maize leaf spot, which is applied to the molecular detection of Curvularia maize leaf spot, and can accurately and rapidly detect Curvularia maize Molecular identification of leaf spot pathogen can also be used to determine whether the diseased corn seedlings with leaf spot and necrotic symptoms are caused by Curvularia maize leaf spot infection. It has great application prospects in field monitoring.

Description

1. Technical field: [0001] The present invention involves fungal disease detection technology in the field of biotechnology, which specifically involves the PCR detection kit and detection method of corn bending leaf spots. 2. Background technology: [0002] Cornbacteria cobiotics is caused by corn curd (Wakker) BOED, causing major hazards in most countries in the world.In 1996, the disease caused severe corn losses in China, and 40 % of the corn production area was infected by the disease.The pathogen is mainly harmful to corn leaves, and it can also harm the leaf sheath and bud leaves.The lesions are soaked in water or pale yellow translucent dots at the beginning, and then expanded into circular, ellipse, shuttle or long strip spots. The shape and size of the diseased spots are divided into 3 categories due to the variety resistanceTransparent halo; ② Intermediate lesions (M): small lesions, 1 ~ 2mm, circular, oval, long strips or irregular shapes, central white or light brown...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12R1/645
Inventor 刘铜侯巨梅左豫虎慕庆峰马柄辰
Owner 贵州益百亿生物科技有限公司
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